Functional analysis of Niemann-Pick disease type C family protein, NPC1a, in Drosophila melanogaster

The Hh pathway is one of the most versatile signaling circuitry reiteratively employed in a variety of developmental contexts (Ingham and McMahon, 2001; Jiang and Hui, 2008; Briscoe and Thérond, 2013). Hh was first discovered in the genetic screens performed in Drosophila melanogaster to uncover the key players during embryonic patterning (Nüsslein-Volhard and Wieschaus, 1980). During embryogenesis, spatially restricted expression of Hh is necessary to regulate downstream targets such as the Drosophila Wnt1 ortholog Wingless (Wg) in neighboring cells (Perrimon et al., 2012). Detailed molecular genetic analysis has also uncovered a role for Hh signaling in patterning the appendages of the adult fly (Strigini and Cohen, 1997). Hh is expressed along the posterior compartment boundary of the wing imaginal disc and activates target genes in the anterior compartment to specify the positional identity of cells across the disc (Tabata and Kornberg, 1994; Basler and Struhl, 1994; Struhl et al., 1997).

The multi-pass transmembrane protein encoded by the segment polarity gene patched (ptc) is the Hh receptor. The binding of Hh ligand to Ptc receptor promotes pathway activation by inhibiting its function. Interestingly, ptc⁻ embryos display ectopic expression of Hh targets irrespective of Hh activity as Ptc suppresses the signal transduction downstream of Hh by inhibiting Smoothened (Smo). Smo, a member of G-protein-coupled receptor (GPCR) superfamily, is absolutely essential for signal transduction downstream of Hh. Consistently, complete loss of smo function eliminates Hh target expression (Alcedo and Noll, 1997; Gallet, 2011). Ptc may participate in transporting lipid moieties that directly or indirectly inhibit Smo activity (Carstea et al., 1997). Consistent with the notion, Ptc protein structure resembles the cholesterol transporter NPC1a.

While Hh primarily functions as a morphogen, it also moonlights as a guidance cue during cell migration in a number of different contexts, including germ cell migration in Drosophila embryos (Deshpande et al., 2001; Charron et al., 2003; Yam et al., 2009). The embryonic gonad of Drosophila is generated by the coalescence of the somatic gonadal precursor cells (SGPs) and the primordial germ cells (PGCs). Mesodermally derived SGPs are specified in three bilateral clusters in parasegments 10-12 (Jaglarz and Howard, 1994; Boyle and Dinardo, 1995; Boyle et al., 1997). By contrast, the PGCs are formed at the posterior pole of the cellular blastoderm. After internalization post-gastrulation, PGCs migrate through the embryo to reach the mesodermal SGPs. The migratory path of PGCs is determined, in part, by the attractive signals generated by the SGPs (Van doren et al., 1998; Santos and Lehmann, 2004a; Deshpande et al., 2017).

Our earlier ‘gain’- and ‘loss’-of-function studies using specific components of Hh pathway have suggested that hedgehog (hh) produced by the SGPs functions as an attractant for the migrating PGCs. First, a subset of germ cells mis-migrates towards an ectopic source of Hh ligand. Confirming the idea that PGCs sense the Hh ligand itself, pattern of PGC migration is affected when embryos are maternally compromised for different members of the Hh signal transduction pathway. Importantly, the phenotypic consequences are distinct and reciprocal, depending on the function of the specific component. Embryos maternally compromised for the positive members, such as smo (smo^m−) or  fused (fu^m−), display scattering of the PGCs across the mesoderm. By contrast, in the embryos derived from the mothers compromised for the inhibitors of the pathway, such as ptc or Pka, PGCs clump precociously. This inappropriate clustering of the PGCs is suggestive of premature activation of the Hh pathway.

Taken together, these data argue that Hh functions as a PGC attractant; however, mechanisms underlying specific potentiation of the SGP-derived Hh are still under investigation. Supporting the possibility, we have shown that a number of well documented regulators of germ cell migration, including Hmgcr, qm and Fpps, also function as components of the hh signaling pathway (Deshpande et al., 2009, 2013). Moreover, our data suggest that Hmgcr helps to mediate the release and/or transport of the Hh ligand emanating from Hh-expressing cells. This is especially relevant as Hmgcr expression is not only involved in the production of an SGP-specific attractant but its expression is also progressively restricted to the SGPs (Van doren et al., 1998; Santos and Lehmann, 2004b; Deshpande and Schedl, 2005).

In addition to the Hmgcr→isoprenoid biosynthetic pathway, other mechanisms are likely to modulate the production and long-distance signaling activity of Hh. The covalent cholesterol modification plays a crucial role in the intracellular sorting of the processed Hh peptide, and its subsequent release and transmission from basolateral membranes (Porter et al., 1996; Mann and Beachy, 2004; Callejo et al., 2011). Unlike mammals, flies are cholesterol auxotroph (Clayton, 1964; Edwards and Ericsson, 1999). Thus, hh signaling would be expected to be sensitive to the levels of dietary cholesterol. The connection between cholesterol, Hh signaling and its potential involvement in germ cell migration is underscored by the discovery of another component of germ cell migration, the ATP-binding cassette (ABC) transporter Mdr49 (Higgins, 1992; Ricardo and Lehmann, 2009). Although it was postulated that Mdr49, which is produced in the mesoderm, assists the release and/or transport of the PGC attractant from the SGPs, these studies did not identify the specific attractant. Employing the ‘loss’- and ‘gain’-of-function strategies, we showed that Mdr49 potentiates the signaling activity of the Hh ligand. Consequently, in Mdr49 mutant embryos, Hh is inappropriately sequestered in the hh-expressing cells. Supporting a role for Mdr49 in providing cholesterol for Hh modification, PGC migration defects in Mdr49 embryos are ameliorated by a cholesterol-rich diet (Deshpande et al., 2016).

Emboldened by these data, we hypothesized that germ cell behavior may depend upon factors directly responsible for cholesterol transmission. Here, we report involvement of a well-documented cholesterol transporter, NPC1a, in germ cell migration and Hh signaling.

Npc1a encodes a membrane-associated protein containing a sterol-sensing domain and is involved in intracellular trafficking of cholesterol (Huang et al., 2005). Mutations in the human ortholog of Npc1a cause a neurodegenerative lysosomal storage disorder called Niemann-Pick type C (NPC) disease (Phillips et al., 2008). Similarly, mutations in its Drosophila homologs, Npc1a and Npc1b, confer a homozygous lethal phenotype during larval stages (Fluegel et al., 2006; Voght et al., 2007). Loss of Npc1a function results in inappropriate accumulation of cholesterol inclusion bodies in neurons, resulting in neuronal degeneration.

To assess whether Npc1a is necessary during germ cell migration, we employed two different mutant alleles: a null allele, Npc1a^57A, and a partial, loss-of-function allele, Npc1a^ep (Fig. 1). In Npc1a mutant embryos, near normal numbers of PGCs are formed that enter and exit the posterior midgut by stage 10 (Ore-R: mean ∼23; n=14 V/S NPC1a: mean ∼24.7; n=12). By stage 13, however, PGCs are not aligned against the SGPs and in several instances they are scattered in the posterior of the embryo (Fig. 1C,D). Embryos from the null allele (Npc1a^57A) show severe germ cell migration defects, as opposed to those derived from the weak allele (∼70% of Npc1a^57A embryos and ∼30% of Npc1a^ep embryos show five or more mis-migrated germ cells). Thus, loss of Npc1a influences germ cell migration adversely. Moreover, Npc1a activity does not seem to be required either for specification or for proper migration of the SGPs. We estimated the total number of SGPs by employing anti-Clift specific staining to mark the SGPs. The average number of SGPs varies between 30 and 34 in the coalesced gonads at stage 14/15 (wild-type mean=30.7, n=8; Npc1a mean=32.6, n=11; UAS Npc1a-RNAi/ twist Gal4 mean=34, n=7).

The larval lethality of homozygous Npc1a mutants can be rescued by raising mutant flies on a cholesterol-rich medium (Fluegel et al., 2006). We therefore wondered whether similar treatment can rescue the embryonic PGC migration defects in the progeny of such mutant adults. Compared with the embryos derived from the Npc1a mutant flies raised on normal diet, those derived from the mutant flies raised on cholesterol-rich diet displayed a less severe mutant phenotype. Approximately 70% of Npc1a mutant embryos typically have extreme germ cell migration defects (5-7+ lost PGCs). By contrast, only 50% of the embryos from Npc1a mutants raised on cholesterol-rich food have extreme germ cell migration defects (Fig. S1B).

To assess whether a long-term feeding method can further improve the extent of rescue, we raised consecutive generations of Npc1a/CyO flies on a cholesterol-rich food. The flies resulting from this approach yielded embryos that exhibited a dramatic rescue (Fig. S1C). Almost 75% of the mutant progeny embryos from mutant adults raised on cholesterol-rich diet (vial-fed) showed negligible germ cell migration defects (0-4 germ cells lost; ∼55% had lost 0-2). Overall, the offspring of Npc1a flies exposed to cholesterol-rich diet exhibit a significant reduction in germ cell migration defects compared with those raised on a normal diet.

We have previously shown that germ cell migration defects induced by the loss of Mdr49 can be substantially ameliorated by raising the flies on a cholesterol-rich diet. Although Mdr49 and Npc1a homozygous mutants display germ cell migration defects, Mdr49/+ or Npc1a/+ embryos demonstrate no significant deviation from wild type. We sought to examine whether Mdr49 and Npc1a mutations show additive or synergistic interactions during germ cell migration. Thus, we crossed Npc1a heterozygous females with Mdr49 heterozygous males, and screened Npc1a/Mdr49 trans-heterozygous embryos for germ cell migration defects (Fig. S2). More than 55% of trans-heterozygous embryos exhibited significant germ cell migration defects (5-7⁺ lost PGCs) when compared with 10% defects observed in heterozygous embryos, indicating that simultaneous partial loss of both Npc1a and Mdr49 is sufficient to disrupt germ cell migration (satisfyingly, similar genetic interaction was also observed between hh and NPc1a mutations; Fig. S2).

Mdr49 is expressed specifically in the mesoderm and twist-GAL4-driven expression of Mdr49 is sufficient to rescue germ cell migration defects in Mdr49 embryos (Ricardo and Lehmann, 2009). The genetic interaction between Mdr49 and Npc1a prompted us to assess a mesodermal requirement of Npc1a. To manipulate its expression in a cell-type-specific manner, we employed the UAS-GAL4-based mis-expression system. Npc1a expression was selectively compromised using twist-GAL4, a mesoderm-specific driver and UAS-Npc1a RNAi. Mdr49-RNAi was used as a positive control. Embryos mesodermally compromised for Npc1a (twi-GAL4/UAS-Npc1a-RNAi) and Mdr49 (twi-GAL4/UAS-Mdr49-RNAi) showed germ cell migration defects by stage 13, unlike twi-GAl4/UAS-GFP-RNAi embryos (Fig. 2A-C). Thus, inactivation of Npc1a in the mesoderm can recapitulate the germ cell migration defects detected in Npc1a embryos.

By contrast, the pattern of germ cell migration in the Npc1a-RNAi X nanos-GAL4 embryos was comparable with that of the twi-GAl4/UAS-GFP-RNAi control. Similarly, germ cell-specific overexpression of Npc1a (nanos-Gal4×UAS-Npc1a) did not influence germ cell count or migration behavior (Fig. 2D). Taken together these data suggest that change in Npc1a levels in a PGC-specific manner does not influence PGC behavior appreciably.

Next, to test whether restoring Npc1a activity can mitigate the effect of Npc1a-RNAi expression on germ cell behavior, we simultaneously overexpressed Npc1a and Npc1aRNAi in the mesoderm. A significant proportion of twist-gal4; UAS-Npc1a/ UAS-Npc1a-RNAi embryos showed near normal germ cell migration, whereas ∼40% of twi-GAL4; UAS-Npc1a-RNAi embryos displayed moderate to severe migration defects (Fig. S3, compare A with B and C). These data establish that mesoderm-specific inactivation of Npc1a is responsible for aberrant germ cell migration and that overexpression of Npc1a in the mesoderm can ameliorate these defects.

Although Npc1a appears to be needed in the mesoderm for PGC migration, it was unclear whether it functions in a cell-autonomous manner. To assess whether Npc1a functions in the same cell type that generates the germ cell attractant, we decided to examine its ability to influence Hmgcr activity. Selective SGP-specific enrichment in Hmgcr expression is instrumental in attracting the PGCs towards them. Supporting its involvement in potentiating the attractant, ectopic expression of Hmgcr induces severe germ cell migration defects. For example, in elav-GAL4 UAS-Hmgcr embryos, where Hmgcr is ectopically expressed in the central nervous system, germ cells are attracted to both the endogenous source of Hmgcr in the mesoderm and the ectopic source of Hmgcr in the central nervous system (Ricardo and Lehmann, 2009; Deshpande et al., 2013). As a result, many PGCs end up migrating towards the central nervous system instead of reaching their correct destination, the SGPs.

We were therefore curious whether reducing Npc1a activity would weaken the strength of the ectopic source of Hmgcr, ultimately resulting in partial suppression of PGC migration defects. Conversely, increasing the expression of Npc1a in the CNS should enhance the migration defects due to potentiation of the attractant(s).

To test this hypothesis, elav-Gal4 UAS-Hmgcr recombinant males were crossed with either homozygous UAS-Npc1a, UAS-Npc1a-RNAi or white¹ (Fig. 3) females. The progeny embryos were assessed for germ cell migration defects. Interestingly, simultaneous ectopic expression of Hmgcr and Npc1a induced modestly enhanced migration defects when compared with control embryos (elav-GAL4 UAS-Hmgcr×w¹). By contrast, the elav-GAL4 UAS-Hmgcr/UAS-Npc1a-RNAi embryos showed significantly reduced germ cell migration defects (Fig. 3). Taken together, these data demonstrate that germ cell migration defects induced by the ectopic Hmgcr expression are sensitive to levels/activity of Npc1a. In this regard, Npc1a appears to behave similarly to Mdr49 and likely is required in cells that synthesize the PGC attractant (Ricardo and Lehmann, 2009).

We have previously shown that Hmgcr and Mdr49 can influence the range/strength of Hh signaling, possibly via enhancing the transmission of Hh ligand. Importantly, such an effect was even observed in Hmgcr^Z− and Mdr49^Z− embryos. (Z⁻ refers to the homozygous mutant embryos derived from the mutant/Balancer stock. Such embryos still have half the level of maternal product.) As Npc1a activity seems to contribute to germ cell migration in conjunction with Mdr49 and hh, we wondered whether Npc1a also contributes to Hedgehog signaling. However, unlike Hmgcr^z− and Mdr49^z− embryos, Npc1a^z− embryos displayed normal levels of Hh target genes (wg, en) in the embryonic ectoderm (Fig. S4). The absence of an obvious phenotype could be due to maternal deposition and stability of the Npc1a protein. We therefore decided to compromise Npc1a levels in the imaginal discs.

In Drosophila wing discs, expression of Hh is restricted to the posterior compartment. Hh organizes patterning of the anterior-posterior compartments of the adult wing blade by inducing the expression of downstream targets such as patched (ptc) and decapentaplegic (dpp) in the anterior compartment. In the absence of hh signaling, these target genes are not activated and there are patterning and growth defects along the anterior-posterior (A-P) axis (Chen and Struhl, 1996; Mullor et al., 1997; Strigini and Cohen, 1997).

To examine whether a reduction in Npc1a results in compromised Hh signaling, we knocked down the expression of Npc1a using a wing pouch-specific nubbin-GAL4 driver in combination with UAS-Npc1a-RNAi. Upon staining the wing discs using anti-Patched antibody, the experimental wing discs (nubbin-GAL4; UAS-GFP) showed a readily detectable reduction in Ptc when compared with the control wing discs (driver alone) (Fig. 4). These data support the claim that Hh signaling is attenuated in wing discs compromised for Npc1a activity. Furthermore, the nubbin-GAL4; UAS-Npc1a-RNAi wing discs also exhibited significant reduction in both patched and dpp transcript levels (Fig. 4F) when compared with the control. As ptc and dpp are biologically relevant targets of Hh morphogen during wing disc patterning, these data strongly support the claim that Hh signaling is attenuated in wing discs compromised for Npc1a activity.

The central region of the Drosophila wing spanning the anterior L3 vein, the posterior L4 vein and the L3-L4 intervein region is directly specified by Hh signaling. The length of the anterior cross vein (ACV) adjoining L3 and L4 veins is highly responsive to changes in either Hh activity or the levels of the downstream target Collier/Knot (Crozatier et al., 2002). To assess whether the observed reduction in Hh signaling (Ptc levels) leads to phenotypic consequences, we examined the wings of adult flies. Indeed, compromising Npc1a in the wing disc proper using nubbin-GAL4 resulted in significant reduction in the length of the ACV (Fig. S5).

In summary, to assess whether PGC migration in Drosophila embryos is sensitive to cholesterol levels, we focused our attention on a cholesterol transporter, NPC1a, and its connection to both Hh signaling and PGC migration. In flies and in mammals, covalent cholesterol modification of the Hh ligand plays a crucial role in the intracellular sorting of the processed Hh peptide, and its subsequent transmission and release from basolateral membranes. Our data indicate that Npc1a likely contributes to generation of sufficient levels of functional cholesterol-modified Hh. Consistently, loss of Npc1a can mimic partial loss of Hh activity in adult wings and also leads to a consistent reduction in transcript levels of canonical Hh target genes. Importantly, involvement of NPC1a in Hh signaling appears to be conserved because in Npc1-deficient mice, subcellular localization of Shh effectors and ciliary proteins is abrogated. Moreover, in NPC1-deficient mouse brains and the human fibroblasts of individuals with NPC1, the aberrant Shh signaling is correlated with diminished cilia length and the total number of ciliated cells (Canterini et al., 2017). Together, these observations support a novel functional connection between Hh signaling and NPC1a activity. Future experiments will determine whether NPC1a is directly responsible for Hh transmission and whether compromised Hh signaling contributes to Niemann-Pick disease symptoms.

Embryo collection and staining were performed essentially as described previously (Deshpande et al., 2013). Vasa (from Paul Lasko, McGill University, Canada) and Hh (from Tom Kornberg, University of California, San Francisco, USA) antibodies are rabbit polyclonal antibodies. Both were used at 1:500 dilution. Engrailed and Wingless antibodies (Developmental Studies Hybridoma Bank) are mouse monoclonal antibodies and were used at 1:10 dilution. β-Galactosidase antibody was either a rabbit polyclonal [MP Biomedicals (previously Cappel), 55976; 1:1000] or a mouse monoclonal antibody (Developmental Studies Hybridoma Bank; 1:10). GFP antibody was a rabbit polyclonal (Torrey Pines Biolabs, ABIN2562810; 1:1000). Secondary antibodies were purchased from Molecular Probes and used at 1:500. For confocal analysis, 40× magnification was used in almost all the analysis, and images were collected using identical settings for the control and experimental samples on a Nikon A1 microscope with GaAsP detectors.

Imaginal discs from third instar larvae were fixed and stained using standard techniques (Callejo et al., 2011). The specific primary antibodies used were: mouse anti-Ptc [1:100; DSHB Drosophila Ptc (Apa 1)-s]. Secondary antibodies were conjugated with Rhodamine Red-X (1:400; Jackson Labs). Images were captured on an A1R confocal microscope (Nikon). Figures were edited using Adobe Photoshop 7.0. Data were analyzed using Nis Elements software.

Fly stocks were raised at 25°C, on standard medium and were primarily obtained from the Drosophila FlyBase (Bloomington, IN, USA). Alleles used in the npc1a experiments are as follows: npcla^57A and npcla⁠. The npcla^ep1 refers to the npcla allele. In the npc1a RNAi ectopic expression experiment, virgin females homozygous for the transgene UAS npc1a RNAi, UAS Mdr49 RNAi or UAS GFP RNAi were crossed with males homozygous for the transgene twist-GAL4 or nanos-GAL4. Gγ1 mutant stocks, Gγ1^N159 and Gγ1^k0817, were obtained from Fumio Matsuzaki (RIKEN Center for Developmental Biology, Kobe, Japan). The Mdr49 mutants and overexpression stocks were obtained from Ruth Lehmann (Skirball Biomedical Institute, NYU, USA). The UAS-RNAi lines specific for Mdr49 (Bloomington, 32405) and Gal4 stocks used for the misexpression studies were from the Bloomington Stock Center (patched-Gal4, UAS-β-galactosidase and hh-Gal4/TM6 Ubx-lacZ). In most experiments, males carrying two copies of the UAS transgene were mated with virgin females carrying two copies of the Gal4 transgene. Embryos derived from the cross were fixed and stained for subsequent analysis. The genotypes of the embryos were unambiguously determined by using the balancer chromosomes marked with either GFP or β-galactosidase. The following stocks were used for analysis of wing discs: UAS-GFP (Bloomington, 1522), UAS-dicer-2; nub-Gal4 (Bloomington, 25754) and UAS npc1a RNAi; UAS npc1a RNAi (VDRC, 42782; Bloomington, 37504). Transgenes were expressed using the Gal4-UAS binary system.

Total RNA was isolated from third instar larval wing discs (∼30 for each sample) of UAS-dicer; nub-Gal4; UAS-GFP (control) and npc1a RNAi KD; UAS-dicer; nub-Gal4/UAS-npc1a RNAi; UAS-npc1a RNAi (experimental) using the QIAGEN RNeasy mini kit. Briefly, wandering third instar larvae were collected, males were selected and dissected in chilled 1×PBS. For each wing disc, the notum was removed. The pouch and hinge tissue were collected in the lysis buffer. Total RNA was extracted as per the manufacturer's instructions. cDNA was made using the high-capacity cDNA reverse transcription kit (Applied Biosystems) using an equal amount of total RNA from each sample. Real-time q-PCR analyses were carried out using the Powersyber Green PCR Master Mix and QuantStudio 12k flex (Applied Biosystems). Rp49 and TBP served as reference genes. Each sample was analyzed in triplicate; the results were confirmed by at least two independent experiments.

Primer sequences used for qPCR were: RP49-F, 5′-TAAGCTGTCGCACAAATGGC-3′; RP49-R, 5′-CCGTAACCGATGTTGGGCAT-3′; TBP-F, 5′-GGCAAAGAGTGAGGACGACT-3′; TBP-R, 5′-GTCGAGGAACTTTGCAGGGA-3′; npc1a-F, 5′-TCATCGAAACAGGACTGCGT-3′; npc1a-R, 5′-ACCGTCAGTTGCCATTTCCT-3′; ptc primer set 1-F, 5′-TACGGAGCTTCTCAGGGCAA-3′; ptc primer set 1-R, 5′-CCAATGGCAGACTCTTGGGT-3′; ptc primer set 2-F, 5′-CACCTCATTCCGTGCTCGAT-3′; ptc primer set2-R, 5′-GAGGCCTGGTATAACGACCG-3′; dpp-F, 5′-GCCAACACAGTGCGAAGTTT-3′; and dpp-R, 5′-GTCGGCGGGAATGCTCTT-3′.

Drosophila food containing cholesterol was prepared as described previously (Fluegel et al., 2006). Cholesterol (Sigma) was used to prepare a stock solution (30 mg/ml) in ethanol. The final concentration was 200 ng/ml. To achieve uniform dispersion of cholesterol, food was mixed thoroughly after cooling it down to 65°C before pouring into either vials or bottles.

Embryos between stages 13 and 15 of a given genotype were analyzed, and the number of germ cells accounted for were the germ cells that showed deviation from the location of the somatic gonadal precursor cells at these stages. For each genotype, the number of embryos examined is indicated in the corresponding graph legend. All staining experiments were repeated three times and statistical significance was estimated using standard two-tailed t-test.

Flies that were ‘cholesterol-fed’ were fed yeast that was mixed with cholesterol, as opposed to just yeast during the time of egg laying and collection. The cholesterol powder was purchased from Sigma and was dissolved in acetone. The yeast paste was mixed with the cholesterol solution (200 µg/ml) to give a final concentration of 200 ng/ml. Flies that were ‘vial-fed’ were fed according to the procedure described by Fluegel et al. (2016). npc1a/+ flies were fed food that contained 200 ng/ml cholesterol for 6 days. These flies were then placed on plates where they ate cholesterol-rich food for egg lay and collection.

For antibodies and stocks, we acknowledge the Developmental Studies Hybridoma Bank, Drs P. Lasko, T. Kornberg, I. Guerrero, R. Lehmann, F. Matsuzaki, E. Olson and the Bloomington Stock Center. We also thank Eric Wieschaus and Trudi Schupbach for helpful discussions and overall encouragement. We thank Dr Gary Laevsky and the Molecular Biology Confocal Microscopy Facility, which is a Nikon Center of Excellence. Gordon Grey provided fly media. We thank Ronit Sionov and Eliran Kadosh for assisting with the qPCR.