Loss of hyx results in NSC overgrowth in the larval central brain

In a clonal screen of a collection of chromosome 3R mutants induced by ethyl methanesulfonate (EMS) [53], we identified 2 new hyrax (hyx) alleles—hyx^HT622 and hyx^w12-46—which produce NSC overgrowth phenotype in the central brain of Drosophila larvae at 96 h after larval hatching (ALH) (Fig 1A and 1B). hyx/CG11990 encodes a highly conserved, 531-amino acid protein that is homologous to mammalian Parafibromin/Cdc73. The hyx^HT622 allele contains a 74-bp deletion from nucleotides 728 (immediately after amino acid 242) to 801, which results in a frameshift mutation and, consequently, the generation of a stop codon at amino acid 248. This likely produces a truncated Hyx protein. The other hyx allele, hyx^w12-46, carries 2-point mutations at nucleotides 1331 (T to G) and 1456 (T to A), which causes amino acid substitutions—Leucine (L) to Arginine (R) at amino acid 444 and Cysteine (C) to Serine (S) at amino acid 486, respectively. As hyx^HT622 and hyx^w12-46 homozygotes are embryonically lethal, we generated MARCM (mosaic analysis with a repressible cell marker) clones [54] to examine the clonal phenotype at larval stages. Hyx protein in the clones was detected using guinea pig anti-Hyx antibodies that we generated against the N-terminal 1–176 amino acids of Hyx. In wild-type control clones, Hyx was predominantly localized to the nuclei of NSCs (S1A and S1B Fig; 0.74-fold in the nuclei, 0.26-fold in the cytoplasm, total intensity:1; n = 15 NSC) and their progeny, but not on mitotic structures (S1A and S1B Fig). In contrast, Hyx was undetectable in 91.3% and 40% of clones generated from hyx^HT622 and hyx^w12-46 alleles, respectively, and was dramatically reduced in the rest of the clones. The intensity of Hyx was significantly reduced to 0.15-fold in hyx^HT622 NSCs (n = 25) and 0.32-fold in hyx^w12-46 NSCs (n = 28), in contrast to 1-fold in control NSCs (S1A–S1C Fig; n = 20). Moreover, we performed western blot with protein extracts from FRT82B, hyx^HT622, and hyx^w12-46 homozygous embryos, as hyx homozygous mutants do not survive to larval stages. Relative protein intensity of Hyx at 24 h after egg laying (AEL) was reduced to 0.31-fold and 0.26-fold in both hyx^HT622 and hyx^w12-46, respectively (S1D and S1E Fig; control, 1-fold). Maternal Hyx might partially contribute to the detected Hyx proteins in these samples. Hyx levels were also significantly reduced in hyx RNAi (RNA interference) in hyx^HT622/+ background to 0.22-fold in third instar larval brains driven by NSC-specific driver insc-Gal4 (S1D and S1E Fig). This western blot result is consistent with our immunofluorescence data that Hyx is dramatically diminished with weak signal in these hyx mutants at the third instar stage (S1A–S1C Fig). Therefore, these observations indicate that hyx^HT622 and hyx^w12-46 are 2 strong loss-of-function alleles.

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Fig 1. Hyx regulates the homeostasis of central larval brain NSCs.

(A) An illustration of 2 hyx EMS alleles. (B) Type I and type II MARCM clones of various genotypes were labeled for Dpn, Ase, and CD8-GFP. Multiple NSCs were observed in hyx-depleted type I and type II MARCM clones. (C) Percentage of clones with ectopic NSCs for genotypes in B. Type I clones: control (FRT82B), n = 61; hyx^HT622, 87%, n = 92; hyx^w12-46, 30.8%, n = 68. Type II clones: control (FRT82B), n = 57; hyx^HT622, 75%, n = 75; hyx^w12-46, 73.3%, n = 66. (D) Average NSC number per clone (with SEM) for genotypes in B. Type I clones: control (FRT82B), 1, n = 61; hyx^HT622, 5.7 ± 1.04, n = 92; hyx^w12-46, 2.8 ± 0.47, n = 68. Type II clones: control (FRT82B), 1, n = 57; hyx^HT622, 6.2 ± 1.17, n = 75; hyx^w12-46, 5.0 ± 1.30, n = 66. (E) NSC lineages from control (UAS-β-Gal RNAi), and hyx RNAi hyx^HT622/+ pupal brains at 10 h APF under the control of insc-Gal4 driver were stained with PH3, Dpn, Ase, and Mira. PH3 and Mira were probed in the same channel. More NSCs positive for PH3 marker were observed in hyx RNAi hyx^HT622/+ papal brains (control, 11.8%, n = 34; hyx RNAi (KK) hyx^HT622/+, 54.8%, n = 31). MARCM clones are outlined by dotted lines. Arrows indicate NSCs. Statistical significances were determined by one-way ANOVA. In D type I, ***p = 0.0005, ns = 0.2582; in D type II, ***p = 0.0006, **p = 0.0019. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. APF, after puparium formation; EMS, ethyl methanesulfonate; Hyx, Hyrax; MARCM, mosaic analysis with a repressible cell marker; Mira, Miranda; NSC, neural stem cell; RNAi, RNA interference.

https://doi.org/10.1371/journal.pbio.3001834.g001

In the central brain of Drosophila larvae, there are at least 2 types of NSCs, both of which divide asymmetrically [55–57]. Each type I NSC generates another NSC and a ganglion mother cell (GMC) that gives rise to 2 neurons, while each type II NSC produces an NSC and a transient amplifying cell (also known as an intermediate neural progenitor or INP), which, in turn, go through a few cycles of asymmetric divisions to produce GMCs [55–58]. In the wild-type control, only 1 NSC is maintained in each of type I or type II MARCM clones (Fig 1B–1D). However, ectopic NSCs were observed in 87% of type I clones and 75% of type II clones generated from the hyx^HT622 allele (Fig 1B). Similarly, supernumerary NSCs were observed in both type I and type II NSC lineages from hyx^w12-46 clones (Fig 1B). Ectopic NSCs per clone were observed in type I hyx^HT622 (5.7) and hyx^w12-46 (2.8) clones and type II hyx^HT622 (6.2) and hyx^w12-46 (5.0) clones (Fig 1D). In addition, knockdown of hyx by 2 independent RNAi lines, under the control of an NSC driver insc-Gal4, led to the formation of multiple NSCs in both type I and type II lineages (S1F and S1G Fig). Moreover, the NSC overgrowth phenotype in hyx^HT622 and hyx^w12-46 mutants was fully rescued by the overexpression of a wild-type hyx transgene (S2A and S2B Fig). Therefore, our finding shows that hyx prevents NSC overgrowth in both type I and type II lineages.

Next, we wondered whether the supernumerary NSCs detected in hyx-depleted larval brains could persist in proliferation in pupal stages. At 10 h after puparium formation (APF), 88.2% of the NSCs in pupal brains were negative for PH3 (Fig 1E and 1F). In contrast, 54.8% of hyx RNAi hyx^HT622/+ NSCs were proliferative and marked by PH3, suggesting that increased number of hyx-depleted NSCs were still actively dividing in early pupal stages. This phenotype might be due to an NSC decommissioning defect or abnormal NSCs that were generated by symmetric divisions acquired proliferative potential. Temporal transcription factors are known to schedule Pros-dependent cell cycle exit of NSCs at the end of larval stages [59]. The abnormal hyx-deficient NSCs often undergo symmetric division, which might result in a disruption of temporal factor transition and, in turn, continued proliferation after pupal formation.

Remarkably, the ectopic NSC phenotype observed in hyx^HT622 larval brains was completely rescued by the overexpression of Parafibromin/HRPT2, the human counterpart of Hyx (S2C Fig). Likewise, the NSC overgrowth phenotype observed in hyx knockdown brains was fully restored by the introduction of human Parafibromin/HRPT2 in both type I and type II NSC lineages (S2D Fig). Therefore, Parafibromin/Hyx appears to have a conserved function in suppressing NSC overgrowth.

Parafibromin/Cdc73 (Hyrax/Hyx in Drosophila) is a component of the Paf1 complex, an evolutionarily conserved protein complex that functions in gene regulation and epigenetics [60–63]. The Paf1 complex also consists of other core subunits Paf1 (Antimeros/atms in Drosophila), Leo1 (Another transcription unit/Atu in Drosophila), Cln three requiring 9 (Ctr9 in Drosophila), and Rtf1 [64,65]. We sought to analyze the function of other components of the Paf1 complex in the larval central brains. Surprisingly, although Ctr9 is required to terminate the proliferation of Drosophila embryonic NSCs [33,66], no ectopic NSCs were observed in ctr9^12P023 type I and type II MARCM clones (S2E Fig). Similarly, knockdown of ctr9, atms, or atu under the control of insc-Gal4 did not generate supernumerary NSCs in either type I or type II lineages in the larval central brains (n = 5 for all). Interestingly, knocking down rtf resulted in a weak ectopic NSC phenotype in type II lineages, without affecting type I NSC lineage development (rtf1 RNAi/BDSC#34586: type II, 31.2%, n = 64 and rtf1 RNAi/BDSC#34850: type II, 7%, n = 43). Therefore, our results suggest that Parafibromin/Hyx might prevent NSC overgrowth independent of Paf1 complex function during Drosophila brain development.

Hyx is essential for the asymmetric division of NSCs

The generation of ectopic NSCs in the absence of Hyx function was not due to INP dedifferentiation, as no type II NSCs were generated in both control and hyx RNAi/V103555 derived INP clones (S3A Fig). hyx expression was efficiently down-regulated in these INP clones upon hyx knockdown (S3B Fig). Next, we assessed whether hyx is required for the asymmetric division of NSCs. In wild-type control metaphase NSCs, apical proteins such as aPKC, Insc, Baz/Par3, Par6, and Pins were localized asymmetrically in the apical cortex (Fig 2A–2F). By contrast, aPKC in hyx^HT622 and hyx^w12-46 metaphase NSCs was completely delocalized from the apical cortex to the cytoplasm (Fig 2A and 2B). Similarly, other apical proteins including Insc, Baz, Par6, and Pins in hyx^HT622 metaphase NSCs were no longer localized asymmetrically in the apical cortex, exhibiting weak or punctate signals in the cytoplasm (Fig 2C–2F).

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Fig 2. Hyx is essential for asymmetric cell division of NSCs.

(A) Metaphase NSCs of control (FRT82B; n = 50) and hyx^HT622 MARCM clones were labeled for aPKC, Mira, and DNA. hyx^HT622, aPKC delocalization, 100%; Mira delocalization, 81.6%; n = 49 for both. (B) Metaphase NSCs of control (FRT82B; n = 45) and hyx^W12-46 MARCM clones were labeled for aPKC, Mira, and DNA. 100% delocalization of aPKC and Mira in hyx^W12-46, n = 50 for both. (C) Metaphase NSCs of control (FRT82B; n = 47) and hyx^HT622 MARCM clones were labeled for Insc, Numb, and DNA. 100% delocalization of Insc and Numb in hyx^HT622, n = 49 for both. (D) Control (FRT82B; n = 46) and hyx^HT622 metaphase NSCs were labeled for Baz and DNA. 100% Baz delocalization in hyx^HT622 (n = 49). (E) Control (FRT82B; n = 50) and hyx^HT622 metaphase NSCs were labelled for Par6 and DNA. 100% delocalization of Par6 in hyx^HT622 (n = 49). (F) Metaphase NSCs from control (FRT82B) and hyx^HT622 were labelled for Pins and DNA. 100% delocalization of Pins in hyx^HT622; n = 49 for both genotypes. Clones were marked by CD8-GFP. (G) Metaphase NSCs from control (FRT82B; n = 48) and hyx^HT622 were labelled for Brat and DNA. 100% Brat delocalization in hyx^HT622 (n = 50). (H) Telophase NSCs from control (UAS-β-Gal RNAi) and hyx RNAi (KK/V103555) under the control of insc-Gal4 driver were labeled for PH3 and CD8-GFP. D1 and D2 indicates NSC and GMC diameter, respectively. (I) Ratio of NSC to GMC diameter (with SD) for genotypes in H. Control: 1.8 ± 0.21 μm, n = 11 NSC; hyx RNAi, 1.2 ± 0.31 μm, n = 4 NSC. (J) Still images of time-lapse imaging of control (G147-GFP/+; n = 15 and S1 Movie) and hyx RNAi (KK/V103555; n = 5 and S2 Movie) NSCs expressing G147-GFP under the control of insc-Gal4 at 48 h ALH. G2 phase, metaphase, anaphase, telophase, and cytokinesis were shown. Statistical significances were determined by unpaired two-tailed Student t test. **p = 0.0011. Scale bar: 5 μm. The underlying data for this figure can be found in the S1 Data. ALH, after larval hatching; aPKC, atypical PKC; Baz, Bazooka; GMC, ganglion mother cell; Hyx, Hyrax; Insc, Inscuteable; MARCM, mosaic analysis with a repressible cell marker; Mira, Miranda; NSC, neural stem cell; Pins, Partner of inscuteable; RNAi, RNA interference.

https://doi.org/10.1371/journal.pbio.3001834.g002

Next, we examined the localization of basal proteins in hyx mutant NSCs. Mira was asymmetrically localized in the basal cortex in 100% of wild-type NSCs during metaphase, but its basal localization was severely disrupted in metaphase NSCs of hyx^HT622 and hyx^w12-46 clones (Fig 2A and 2B). Similarly, Numb and Brat lost their asymmetric basal localization and were observed in the cytoplasm in metaphase NSCs of hyx^HT622 clones (Fig 2C and 2G).

Similarly, hyx knockdown by 2 independent RNAi lines disrupted NSC apicobasal polarity. aPKC, Par6, Baz, Insc, and Pins were delocalized from the apical cortex in all metaphase NSCs of both hyx RNAi lines (S2C–S2I Fig). In addition, the localization of Mira, Numb, and Brat proteins at the basal cortex was also disrupted in metaphase NSCs upon hyx knockdown (S2C–S2E and S2I Fig). Taken together, our data indicate that Hyx orchestrates NSC polarity by regulating the proper localization of both apical and basal proteins.

To examine the daughter cell size asymmetry, we measured the ratio of NSC to GMC diameter at telophase. Cells were outlined by a membrane marker CD8-GFP driven by insc-Gal4. The NSC-to-GMC diameter ratio in hyx RNAi telophase NSCs was significantly reduced to 1.2 (Fig 2H and 2I; n = 4) compared with 1.8 (n = 11) in control telophase NSCs. These data indicate that hyx depletion disrupts daughter cell size asymmetry of the NSCs. Given that polarization of NSCs is an essential prerequisite for their asymmetric division, we sought to investigate whether hyx depletion could result in the symmetric division of NSCs. To this end, we took advantage of a microtubule-binding protein Jupiter-GFP (also known as G147-GFP), which is controlled under its endogenous promoter [67]. In live, whole-mount larval brains that expressed G147-GFP, control NSCs always divided asymmetrically to produce 2 daughter cells with distinct cell sizes (Fig 2J and S1 Movie). By contrast, all NSCs in hyx RNAi divided symmetrically to generate 2 daughter cells with similar cell sizes (Fig 2J and S2 Movie). These observations indicate that hyx-depleted NSCs divide symmetrically, leading to NSC overgrowth. However, the allograft transplantation of hyx^HT622 (n = 29 host) and hyx^w12-46 (n = 30 host) homozygous clones and hyx RNAi (n = 30 host) larval brain cells did not induce tumors. Perhaps hyx-depleted cells are small in size and with altered cell fate and have reduced cell growth, thus unable to expand in this tumor assay.

The protein levels of the phosphorylated Akt (Ser505) (P-Akt) were unaffected in hyx RNAi NSCs as compared with that in control NSCs (S4A and S4B Fig; 1.0-fold versus 1.02-fold). In addition, protein levels of a cell cycle regulator String (Stg)/Cdc25 in hyx RNAi NSCs were unaffected (S4C and S4D Fig; 1.01-fold versus 1.0-fold in control). Similarly, microtubule star (Mts), the PP2A catalytic subunit C, was unaffected in hyx^HT622 NSCs (S4E and S4F Fig; intensity normalized against Dpn: 0.89 in control versus 0.78 in hyx^HT622). These data suggest that Hyx does not seem to regulate PI3K/Akt pathway, Cdc25, or Mts in NSCs.

Hyx maintains interphase microtubule asters and the mitotic spindle

When analyzing the asymmetric division, we noticed that hyx-depleted NSCs formed shorter mitotic spindles. The mean spindle length in control metaphase NSCs was 9.81 ± 0.94 μm, whereas in hyx RNAi (V103555) NSCs, the mean spindle length was dramatically shortened to 6.84 ± 1.46 μm (Fig 3A and 3B). Consistent with this observation, spindle lengths in metaphase hyx^HT622 and hyx^w12-46 NSCs were much shorter than that in the control (Fig 3C and 3D). When normalized against the cell diameter of NSCs, the mitotic spindle length was still significantly shortened in hyx-depleted NSCs. The relative spindle length was significantly shortened to 0.74-fold in hyx RNAi NSCs (Fig 3E; n = 15 NSC) compared with 0.82-fold in control NSCs (n = 32 NSC). Similarly, the ratio of spindle length to cell diameter was significantly reduced to 0.70-fold in both hyx^HT622 (n = 13 NSC) NSCs and hyx^w12-46 NSCs (n = 21 NSC) when compared with 0.79-fold in control NSCs (Fig 3F; n = 22 NSC).

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Fig 3. Hyx is required for the formation of the microtubule aster and the mitotic spindle in NSCs.

(A) Metaphase NSCs of control (UAS-β-Gal RNAi) and hyx RNAi (KK/V103555) under the control of insc-Gal4 were labelled for α-tub and DNA. (B) Quantification of spindle length (with SD) observed in A. Spindle length: control, 9.81 ± 0.94 μm, n = 29; hyx RNAi, 6.84 ± 1.46 μm, n = 40. (C) NSCs of control (FRT82B), hyx^HT622, and hyx^W12-46 MARCM clones were labelled for α-tub and DNA and CD8-GFP. (D) Quantification of spindle length (with SD) observed in C. Spindle length: control, 9.70± 1.15 μm, n = 21; hyx^HT622, 6.13 ± 1.22 μm, n = 18; hyx^W12-46, 7.43 ± 1.25 μm, n = 18. (E) Ratio of spindle length to NSC diameter (with SD) for genotypes in A. Control: 0.82 ± 0.08, n = 32; hyx RNAi, 0.74 ± 0.12, n = 15. (F) Ratio of spindle length to NSC diameter (with SD) for genotypes in B. Control: 0.79 ± 0.06, n = 22; hyx^HT622, 0.70 ± 0.10, n = 13; hyx^W12-46, 0.70 ± 0.10, n = 21. (G) Interphase NSCs from control (FRT82B; n = 11), hyx^HT622 (n = 24), and hyx^w12-46 (n = 29) MARCM clones were labelled for α-tub, Asl, GFP, and PH3. NSCs failed to form a microtubule aster in 79.2% of hyx^HT622 and 34.5% of hyx^w12-46 clones. The rest of them formed weak microtubule asters. (H) The immunofluorescence intensity of astral microtubule (with SEM) labelled by α-tub for genotypes in G. Control: 100.7 ± 12.77, n = 7; hyx^HT622, 14. 7 ± 19.12, n = 23; hyx^W12-46, 27.6 ± 32.56, n = 23. Cell outlines are indicated by white dotted lines. Arrows indicate the centrosomes. Statistical significances were determined by unpaired two-tailed Student t test in B. One-way ANOVA with multiple comparison were performed in D, F, and H. ****p < 0.0001 for B, D, F; **p = 0.0065 for E; In F, **p = 0.0045 for hyx^HT622 and **p = 0.0017 for hyx^W12-46. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. Asl, Asterless; α-tub, α-tubulin; Hyx, Hyrax; MARCM, mosaic analysis with a repressible cell marker; NSC, neural stem cell; RNAi, RNA interference.

https://doi.org/10.1371/journal.pbio.3001834.g003

These observations prompted us to examine whether Hyx is important for microtubule assembly in NSCs. We sought to determine whether Hyx regulates the formation of microtubule asters in interphase NSCs. A wild-type interphase NSC forms 1 major microtubule aster marked by α-tubulin (α-tub). Asters are assembled by the microtubule-organizing center (MTOC), also known as centrosomes, of cycling NSCs labeled by a centriolar protein called Asterless (Asl; Fig 3G). Strikingly, the vast majority of interphase NSCs in hyx^HT622 and hyx^w12-46 clones either failed to organize a microtubule aster or formed weak microtubule asters (Fig 3G). The astral microtubule intensity marked by α-tub was dramatically reduced to 14.7 (a.u.) and 27.6 (a.u.) in hyx^HT622 (n = 23 NSC) and hyx^w12-46 NSCs (n = 23 NSC), respectively, significantly lower than 100.7 (a.u.) in control NSCs (Fig 3H; n = 7 NSC). Likewise, in hyx knockdown clones, 50% of NSCs failed to form microtubule asters and 40.9% of NSCs only assembled weak microtubule asters during interphase (S5A Fig). Overall, we show that Hyx is important for the formation of interphase microtubule asters.

The shortened mitotic spindles and defects in the assembly of microtubule asters upon hyx depletion suggested that Hyx might regulate microtubule growth in dividing NSCs. To this end, we performed a microtubule regrowth assay by “cold” treatment of larval brains on ice—for efficiently depolymerizing microtubules in NSCs—followed by their recovery at 25°C to allow microtubule regrowth in the course of time. In both control and hyx RNAi interphase NSCs treated with ice (t = 0), no astral microtubules were observed and only weak residual microtubules labelled by α-tub remained at the centrosome (Fig 4A). The centrosomes in 76.3% of these hyx RNAi interphase NSCs were absent or much smaller in size, suggesting that the MTOC was compromised upon hyx depletion (Fig 4A). In control interphase NSCs, robust astral microtubules were observed around the centrosome, at various time points following recovery at 25°C (Fig 4A). By contrast, the vast majority of hyx RNAi interphase NSCs reassembled scarce microtubule bundles without detectable MTOCs, even 120 s after recovery at 25°C (Fig 4A).

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Fig 4. Hyx promotes microtubule growth in NSCs.

(A) Control (UAS-β-Gal RNAi) and hyx RNAi (KK/V103555) interphase NSCs were labelled for α-tub and DNA at various time points after recovery from treatment on ice in microtubule regrowth assay. At 0 s (t = 0; ice treatment), no astral microtubules around the centrosome/MTOC were observed in all control (n = 74) and hyx RNAi NSCs (n = 63). At all subsequent time points following the recovery, all control NSCs assembled robust astral microtubules (t = 30 s, n = 31; t = 60 s, n = 11; t = 90 s, n = 27; t = 120 s, n = 26). Astral microtubules were lost in hyx RNAi: t = 30 s, 71.9%, n = 43; t = 60 s, 85.7%, n = 7; t = 90 s, 91.2%, n = 46; t = 2 min, 86.3%, n = 63. (B) The immunofluorescence intensity of astral microtubules (with SEM) marked by α-tub for various time points in A. Control: t = 0, 0.5 ± 0.13, n = 19; t = 30 s, 47.6 ± 5.01, n = 22; t = 60 s, 78.4 ± 0.13, n = 12; t = 90 s, 60.4 ± 4.58, n = 16; t = 2 min, 50.8 ± 4.65, n = 15; t = 5 min, 66.8 ± 7.27, n = 12. hyx RNAi: t = 0, 0.7 ± 0.22, n = 27; t = 30 s, 17.6 ± 1.73, n = 35; t = 60 s, 27.8 ± 3.27, n = 10; t = 90 s, 31.3 ± 2.60, n = 39; t = 2 min, 28.9 ± 2.04, n = 42; t = 5 min, 39.0 ± 7.07, n = 11. RNAi was controlled by insc-Gal4. (C) Control (UAS-β-Gal RNAi) and hyx RNAi (KK/V103555) metaphase NSCs were labelled for α-tub and DNA at various time points after recovery from treatment on ice in microtubule regrowth assay. Control: t = 0, n = 52; t = 30 s, n = 19; t = 60 s, n = 9; t = 90 s, n = 41; hyx RNAi, t = 0, n = 28. Scarce spindle microtubule mass assembled in hyx RNAi: t = 30 s, 91.7%, n = 18; t = 60 s, 66.7%, n = 3; t = 90 s, 69.7%, n = 19; t = 2 min, 85.4%, n = 11. RNAi was controlled by insc-Gal4. (D) The immunofluorescence intensity of astral microtubules (with SEM) marked by α-tub for various time points in C. Control: t = 0, 1.4 ± 0.32, n = 30; t = 30 s, 49.4 ± 5.53, n = 26; t = 60 s, 52.3 ± 5.62, n = 17; t = 90 s, 68.2 ± 6.43, n = 21; t = 2 min, 68.8 ± 6.80, n = 11; t = 5 min, 68.3 ± 4.97, n = 28. hyx RNAi: t = 0, 0.8 ± 0.23, n = 30; t = 30 s, 18.0 ± 3.43, n = 15; t = 60 s, 25.3 ± 2.59, n = 14; t = 90 s, 44.7 ± 5.52, n = 13; t = 2 min, 27.8 ± 4.08, n = 15; t = 5 min, 46.3 ± 6.18, n = 16. Cell outlines are indicated by the white-dotted lines. One-way ANOVA with multiple comparison were performed in B and D. In B, ns > 0.9999, ****p < 0.0001 and ***p = 0.0003; in D, ns > 0.9999, ****p < 0.0001, **p = 0.0068 for t = 60 s, **p = 0.0068 for t = 90 s, and **p = 0.0034 for t = 5 min. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. α-tub, α-tubulin; Hyx, Hyrax; MTOC, microtubule-organizing center; NSC, neural stem cell; RNAi, RNA interference.

https://doi.org/10.1371/journal.pbio.3001834.g004

Next, we examined microtubule regrowth in mitotic NSCs. Upon treatment with ice (t = 0), spindle microtubules were effectively depolymerized with residual microtubules marking the centrosomes/spindle poles, in all control and hyx RNAi metaphase NSCs (Fig 4B). Consistent with poor centrosome assembly during interphase, the centrosomes of 98.0% of hyx RNAi metaphase NSCs were deformed with irregular shapes (Fig 4B). In all control metaphase NSCs, intense spindle microtubules were reassembled around centrosomes and chromosome mass from as early as 30 s following recovery at 25°C; the mitotic spindle completely reformed at 2 min following recovery (Fig 4B). In contrast, the majority of hyx RNAi metaphase NSCs assembled scarce spindle microtubule mass following recovery; at 2 min after recovery, only 14.6% of metaphase NSCs formed mitotic spindles, which were still shorter and thinner than the spindles formed in the control NSCs. MTOCs remained weak or absent in these hyx-depleted NSCs (Fig 4B). Quantification of microtubule intensity suggested that in all of the time points (except for t = 0), microtubule intensity in hyx-depleted NSCs at both interphase and metaphase were significantly reduced compared with the control (Fig 4B and 4D). Taken together, we propose that Hyx plays a central role in the formation of interphase microtubule asters and the mitotic spindle by promoting microtubule growth in NSCs.

Hyx is required for centrosome assembly in dividing NSCs

Since we had demonstrated that Hyx promotes microtubule growth and interphase aster formation in NSCs, we wondered whether Hyx regulates the assembly of the centrosomes, the major MTOC in dividing cells. Each centrosome is composed of a pair of centrioles surrounded by pericentriolar material (PCM) proteins. Centriolar proteins Spindle assembly abnormal 4 (Sas-4), Anastral spindle 2 (Ana2), and Asl are essential for centriole biogenesis and assembly [24,68–71]. In control interphase and metaphase NSCs, Sas-4 was always observed at the centrosomes overlapping with Asl (S5B and S5C Fig). In response to hyx knockdown, the localization of Sas-4 and Asl seemed unaffected at the centrosomes in both interphase and metaphase NSCs (S5B and S5C Fig).

Wild-type NSCs typically contained 2 Asl-positive centrioles at both interphase and metaphase (S5D Fig; n = 21 and n = 20, respectively), as the centrioles are duplicated early in the cell cycle. Interestingly, in hyx^HT622 MARCM clones, multiple centrioles labelled by Asl were seen at metaphase NSCs (S5D Fig; 28.1%, n = 32) but not at interphase NSCs (S5D Fig; n = 20). This observation suggests that centriole fragmentation might occur in hyx-depleted mitotic cells. Meanwhile, we found cytokinesis delay in 39.1% of hyx^HT622 clones (S5E Fig). However, no giant, polyploidy cells were observed for hyx-depleted brains, implying that cytokinesis delay is unlikely the primary cause of multiple centriole phenotype in these cells. Consistent with these observations, multiple centrioles were detected in 52.0% of S2 cells upon dsRNA treatment against hyx (S5F and S5G Fig), which was significantly higher than what was observed in control cells (S5F and S5G Fig; 25.1%).

Major PCM proteins γ-tubulin (γ-tub) and centrosomin (Cnn, a CDK5RAP2 homolog) are essential for microtubule nucleation and anchoring. γ-tub is a component of the γ-tubulin ring complex (γ-TURC), which is the major microtubule nucleator in dividing cells, including NSCs [72,73]. In control interphase and metaphase NSCs, robust γ-tub was detected at the centrosomes and was observed to colocalize with Asl (S6A Fig). By contrast, γ-tub was absent or significantly reduced at the centrosomes in 95.7% of hyx^HT622 and 54.8% of hyx^w12-46 interphase NSCs (S6A Fig); the fluorescence intensity of γ-tub was dramatically decreased in these NSCs (S6A and S6C Fig). In addition, γ-tub was strongly diminished at the centrosomes in 93.1% of hyx^HT622 and 70.8% of hyx^w12-46 metaphase NSCs (S6B Fig). The fluorescence intensity of γ-tub protein dropped to 0.17-fold in hyx^HT622 and 0.53-fold in hyx^w12-46 NSCs, respectively (S6B and S6C Fig). Likewise, γ-tub was strongly reduced at the centrosomes in 90.5% of interphase and 81.3% of metaphase NSCs upon hyx RNAi knockdown (S6D and S6E Fig); moreover, the fluorescence intensity of γ-tub was dramatically decreased at the centrosomes in these NSCs (S6D–S6F Fig).

Next, we examined the localization of Cnn, another essential PCM component. During interphase, 98.0% of control NSCs had intense Cnn signal at the centrosomes marked by Asl (S6G and S6I Fig). By contrast, Cnn was barely detectable at the centrosomes in 84.8% of hyx^HT622 and 58.3% of hyx^w12-46 (n = 12) interphase NSCs (S6G and S6I Fig). Consistent with this observation, Cnn levels were significantly diminished at the centrosomes in 72.4% of hyx^HT622 and 66.7% of hyx^w12-46 (n = 18) metaphase NSCs (S6H and S6I Fig). Similarly, Cnn levels were dramatically reduced from the centrosomes in 92.6% of interphase NSCs upon hyx knockdown (S6J and S6L Fig). In metaphase NSCs with hyx knockdown, Cnn intensity at the centrosomes significantly dropped in 46.7% of NSCs (S6K and S6L Fig). Our observations indicate that Hyx ensures the recruitment of PCM proteins γ-tub and Cnn to the centrosomes in both interphase and mitotic NSCs.

Centrosomal localization of Msps, D-TACC, and Polo is dependent on Hyx function in NSCs

Msps is an XMAP215/ch-TOG family protein and a key microtubule polymerase that controls microtubule growth and asymmetric division of NSCs in Drosophila larval central brains [44,74]. In control interphase NSCs, Msps colocalized with Asl at the centrosomes (S7A Fig). However, during interphase, Msps was delocalized from the centrosomes in 86.4% of hyx^HT622 and 47.1% of hyx^w12-46 NSCs (S7A and S7C Fig). Likewise, during metaphase, Msps was nearly absent at the centrosomes in 87.5% of hyx^HT622 and 53.3% of hyx^w12-46 NSCs (S7B and S7C Fig). Moreover, Msps was undetectable at the centrosomes in the majority of interphase NSCs upon hyx knockdown (S7D and S7F Fig). In metaphase NSCs, Msps was detected at the centrosomes only in 16.4% of hyx RNAi NSCs (S7E and S7F Fig).

As the efficient centrosomal localization of Msps depends on D-TACC, a microtubule-binding centrosomal protein [74], we wondered whether D-TACC localization in NSCs requires Hyx function. In all control interphase NSCs, D-TACC was concentrated at the centrosomes marked by Asl (S7G Fig). Remarkably, during interphase, D-TACC was absent from the centrosomes in 92.0% of hyx^HT622 (S7G Fig) and 81.8% of hyx^W12-46 NSCs (n = 22). Similarly, in metaphase NSCs, D-TACC was undetectable at the centrosomes in 95.2% of hyx^HT622 NSCs (S7H Fig) and 80.0% of hyx^w12-46 NSCs (n = 15). Likewise, D-TACC was apparently undetectable at the centrosomes in the majority of interphase and metaphase NSCs upon hyx knockdown (S7J and S7K Fig). The fluorescence intensity of D-TACC was significantly decreased in hyx-depleted NSCs (S7G–S7L Fig). Taken together, our results suggest that Hyx is essential for the centrosomal localization of Msps and D-TACC in cycling NSCs.

As Polo, another key centrosomal protein, is critical for the assembly of interphase microtubule asters and asymmetric cell division [27,75], we tested whether Hyx regulates the localization of Polo at the centrosomes. In control interphase NSCs, Polo was strongly detected at the centrosome marked by Asl (Fig 5A). In contrast, Polo was almost completely absent in 86.8% of hyx^HT622 and 58.8% of hyx^w12-46 interphase NSCs (Fig 5A). Furthermore, in control metaphase NSCs, Polo mainly appeared on the centrosomes and kinetochores and weakly on the mitotic spindle (Fig 5B). However, 80.0% of hyx^HT622 and 75% of hyx^w12-46 metaphase NSCs lost Polo loci, and the remaining NSCs only had a weak Polo signal (Fig 5B). Similarly, upon hyx knockdown, Polo was almost completely lost from the centrosomes in 84.6% of interphase NSCs and 78.3% of metaphase NSCs (S8A and S8B Fig). The fluorescence intensity of Polo was significantly reduced at the centrosomes in hyx-depleted interphase and metaphase NSCs (Figs 5C and S8C).

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Fig 5. Hyx regulates the localization of centrosomal proteins Polo and AurA in NSCs.

(A) Interphase NSCs from control (FRT82B) and hyx^HT622 MARCM clones were labelled for Polo, Asl, GFP, and PH3. Polo delocalization/reduction: control, 5.8%, n = 52; hyx^HT622, 86.8%, n = 38; hyx^w12-46, 58.8%, n = 34. (B) Metaphase NSCs of control (FRT82B), hyx^HT622 and hyx^w12-46 MARCM clones were labelled for Polo, Asl, GFP, and PH3. Polo delocalization/reduction: control, 0%, n = 20; hyx^HT622, 80.0%, n = 10; hyx^w12-46, 75.0%, n = 12. (C) Quantification graph of the fold change of Polo intensity (with SD) in NSCs from A and B. Interphase: control, 1-fold, n = 41; hyx^HT622, 0.12 ± 0.11-fold, n = 38; hyx^w12-46, 0.53 ± 0.25-fold, n = 34. Metaphase: control, 1-fold, n = 20; hyx^HT622, 0.10 ± 0.12-fold, n = 10; hyx^w12-46, 0.46 ± 0.32-fold, n = 12. (D) Interphase NSCs from control (FRT82B; n = 36) and hyx^HT622 (n = 39) MARCM clones were labelled for AurA, Asl, GFP, and PH3. (E) Metaphase NSCs of control (FRT82B; n = 22) and hyx^HT622 (n = 18) MARCM clones were labelled for AurA, Asl, GFP, and PH3. (F) Quantification graph of the fold change of AurA intensity (with SD) in NSCs from D and E. Interphase: control, 1-fold, n = 36; hyx^HT622, 0.11 ± 0.04-fold, n = 39; hyx^w12-46, 0.23 ± 0.04-fold, n = 20. Metaphase: control, 1-fold, n = 22; hyx^HT622, 0.43 ± 0.11-fold, n = 18; hyx^w12-46, 0.79 ± 0.18-fold, n = 8. Cell outlines are indicated by white-dotted lines. Arrows point at the centrosome in A-E. Statistical significances were determined two-way ANOVA with multiple comparison were performed in C and F. In C, **p = 0.0044, ns = 0.0634; in F, **p = 0.0040, *p = 0.0382. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. Asl, Asterless; AurA, Aurora-A; Hyx, Hyrax; MARCM, mosaic analysis with a repressible cell marker; NSC, neural stem cell.

https://doi.org/10.1371/journal.pbio.3001834.g005

Centrosomal protein AurA inhibits NSC overgrowth and regulates centrosome functions by directing the centrosomal localization of D-TACC and Msps [35,76]. We sought to examine whether the centrosomal localization of AurA is dependent on Hyx. AurA is clearly observed at the centrosomes marked by Asl in control interphase and metaphase NSCs (Fig 5D and 5E). Remarkably, AurA was nearly undetectable at the centrosomes in 87.2% of hyx^HT622 and 75.0% hyx^w12-46 interphase NSCs (Fig 5D). The fluorescence intensity of AurA decreased to 0.11-fold in hyx^HT622 and 0.23-fold in hyx^w12-46 interphase NSCs (Fig 5D and 5F). Likewise, AurA levels were significantly reduced in 88.9% of hyx^HT622 and 50% of hyx^w12-46 metaphase NSCs (Fig 5E and 5F). Furthermore, AurA was diminished in 100% of interphase NSCs and 56.7% of metaphase NSCs upon hyx knockdown (S8D–S8F Fig). Taken together, our data show that Hyx plays an essential role in centrosome assembly and functions by recruiting major centrosomal proteins to the centrosomes in NSCs.

The disruption of NSC polarity and centrosome assembly is a direct consequence of hyx depletion, but not aging

To rule out the possibility that the disruption of NSC polarity and centrosome assembly was due to consequence of aging in late larval stages, we examined NSC polarity proteins and centrosomal proteins at 24 h ALH, a time point when NSCs exit quiescence and reenter the cell cycle [77]. At 24 h ALH, Hyx was dramatically lost in NSCs upon knocking down hyx by RNAi under the control of insc-Gal4 driver in hyx^HT622/+ background, suggesting an efficient knockdown of Hyx (S9A Fig). In hyx RNAi hyx^HT622/+ at 24 h ALH, aPKC was delocalized in 96.8% of NSCs and Mira in 91.9% of NSCs, compared with a control that both aPKC and Mira formed proper crescent in all metaphase NSCs (S9B Fig). Likewise, centrosome protein γ-tub was severely reduced at the centrosomes in 82.4% of interphase NSCs and 85.0% of metaphase NSCs from hyx RNAi hyx^HT622/+ (S9C–S9E Fig). Similarly, Polo was largely delocalized from the centrosomes in NSCs from hyx RNAi hyx^HT622/+, which led to a significant decrease of Polo protein levels at the centrosomes in both interphase and metaphase NSCs (S9F–S9H Fig).

In addition, in late larval stages, hyx RNAi hyx^HT622/+ showed a stronger NSC overproliferation phenotype than that observed in hyx knockdown alone (Fig 1B and 1C); 84.3% of type I lineages and 93.3% of type II lineages with multiple NSCs were observed in hyx RNAi hyx^HT622/+ compared with the control with a single NSC per lineage (S10A Fig). Moreover, Hyx protein was diminished in 89.1% of hyx RNAi hyx^HT622/+ NSCs, while it was strongly detected in the control (S10B Fig). Consistent with these observations, strong reduction of γ-tub and Polo protein levels at the centrosomes was observed in both interphase and metaphase NSCs from hyx RNAi hyx^HT622/+ (S10C–S10H Fig).

Taken together, the disruption of NSC polarity and centrosome assembly is a direct consequence of hyx loss of function instead of aging.

Hyx is required for centrosome assembly in S2 cells in vitro

To investigate whether Hyx plays a role in centrosome assembly in nonneuronal cells, we knocked down hyx in S2 cells by dsRNA treatment. We found that a centriolar protein, Ana2, remained localized at the centrosomes in metaphase cells (S8G Fig). This suggests that Hyx is not essential for the localization of centriolar proteins in both S2 cells and NSCs. Next, we examined the localization of other centrosomal proteins in S2 cells. Remarkably, D-TACC intensity was significantly decreased at the centrosomes, upon hyx knockdown, in metaphase S2 cells (Fig 6A and 6B). Consistent with these observations, the intensity of α-tub was also decreased by 0.65-fold on mitotic spindles (Fig 6D and 6E). These in vitro data support our observations in the larval brain and indicate that Hyx regulates microtubule growth and the localization of centrosomal proteins. Polo is undetectable in interphase S2 cells, unlike its robust localization in NSCs during the interphase. Consistent with our in vivo observations, we found that the overall intensity of Polo was significantly reduced to 0.67-fold in the dividing metaphase cells upon hyx knockdown (Fig 6A and 6C). Also, γ-tub intensity at the centrosomes marked by Ana2 was similar to that observed in the control (S8G and S8H Fig). The different observations in S2 cells and larval brains are likely due to incomplete depletion of hyx in S2 cells and/or different underlying mechanisms in vitro.

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Fig 6. Hyx is required for the recruitment of centrosome-related proteins and directly regulates their expression in vitro.

(A) Metaphase cells from ds-egfp-treated S2 cells and ds-hyx-treated S2 cells were labeled for DTACC, Polo, Asl, and DNA. (B) Quantification graph of the fold change of DTACC intensity (with SD) in S2 cells from A. ds-egfp, 1-fold, n = 66; ds-hyx, 0.75 ± 0.05-fold, n = 69. (C) Quantification graph of the fold change of overall Polo intensity (with SD) in S2 cells from A. ds-egfp, 1-fold, n = 77; ds-hyx, 0.67 ± 0.08-fold, n = 81. (D) Metaphase S2 cells treated with ds-egfp and ds-hyx were labeled for α-tub, Asl, and DNA. (E) Quantification graph of the fold change of α-tub intensity in S2 cells from D. ds-egfp, 1-fold, n = 83; ds-hyx, 0.65 ± 0.10-fold, n = 88. (F) Spinning disc super-resolution imaging of Cnn, γ-tub, and Asl in metaphase S2 cells treated with ds-egfp (Cnn, n = 64; γ-tub, n = 55) and ds-hyx (Cnn, n = 58; γ-tub, n = 58). (G) Quantification graph of the percentage (with SD) of metaphase S2 cells forming “doughnut-like” shape of Cnn and γ-tub in F. ds-egfp: Pattern of Cnn, Normal, 94.9% ± 5.60%; abnormal, 5.1 ± 5.60%. ds-hyx: Pattern of Cnn, Normal, 48.7 ± 21.86%; abnormal, 51.3 ± 21.86%. ds-egfp: Pattern of γ-tub, Normal, 92.8 ± 8.57%; abnormal, 7.2 ± 8.57%. ds-hyx: Pattern of γ-tub, Normal, 46.6 ± 22.41%; abnormal, 53.4 ± 22.41%. (H) Location of primer pairs used for ChIP-qPCR on the promoter of genes in I. The schematic represents the polo gene, with the arrow indicating the upstream fragment distance from the TSS and the center nucleotide position of the primer pair is given and AAA showing the approximate location of the cleavage and polyadenylation site. (I) Quantification graph of ChIP-qPCR for detecting occupancy by Hyx on various genes in S2 cells, with an intergenic region at 5 kb downstream of the numb gene as a negative control and orb2 as a positive control. After normalizing against “Pre-serum,” fold enrichment from “Pre-serum” was taken as 1-fold for all primer sets. Fold enrichment (with SD) in “Hyx”: negative control, 1.37 ± 0.18-fold; positive control orb2, 2.94 ± 1.24-fold; polo (−587), 2.95 ± 1.47-fold; polo (−224), 2.63 ± 1.68-fold; aurA, 2.85 ± 1.77-fold; numb, 1.64 ± 0.31-fold. Minimum of 3 biological replicates were performed. (J) Luciferase assay in S2 cells shows an increase of the pGL3-luciferase reporter coupled with polo promoter (poloPro, 643 bp of sequences upstream of TSS) by endogenous hyx expression. Vector (pAFW)+pGL3-basic: 1.0 ± 0.28; Vector (pAFW)+ poloPro: 122 ± 6.05. The relative luciferase activity was normalized to Renilla luciferase activity. (K) Luciferase assay in S2 cells shows down-regulation of the pGL3-luciferase reporter coupled with poloPro ds-hyx treatment. The relative luciferase activity was normalized to Renilla luciferase activity. ds-egfp: 1-fold; ds-hyx: 0.5 ± 0.13-fold. (L) Luciferase assay in S2 cells shows no consistent alterations on the pGL3-luciferase reporter coupled with actin5c promoter between ds-egfp and ds-hyx treatment. The relative luciferase activity was normalized to Renilla luciferase activity. ds-egfp: 1-fold; ds-hyx: 0.2 ± 0.51-fold. (M) Quantification graph of RT-qPCR analysis in 48 h ALH brains from control (UAS-β-gal RNAi; UAS-β-gal RNAi) and hyx RNAi with UAS-Dicer2 driven by actin5C-Gal4. Minimum 3 repeats were conducted. After normalization against control (with SD): control, 1-fold; hyx, 0.23 ± 0.05-fold; polo, 0.33 ± 0.08-fold; aurA, 0.24 ± 0.07-fold; γ-tub, 0.59 ± 0.20-fold; cnn, 0.68 ± 0.17-fold; msps, 0.86 ± 0.15-fold; tacc, 0.45 ± 0.15-fold. (N) 48 h ALH larval brains from control (UAS-β-Gal RNAi; UAS-β-Gal RNAi) and hyx knockdown with UAS-Dicer2 (hyx RNAi; UAS-Dicer2 RNAi) under the control of actin5C-Gal4 were labelled with Hyx, Dpn, and Phalloidin. (O) Quantification graph for (N) showing the fluorescence intensity of Hyx throughout the whole brain and in the NSCs, respectively. Overall Hyx intensity in control: 41.5 ± 13.25, n = 34 ROI (region of interest excluded neuropil region) from 10 BL; hyx RNAi: 19.7 ± 8.99, n = 36 ROI from 8 BL. Hyx intensity in NSCs in control: 107.1 ± 29.18, n = 96 NSCs; hyx RNAi: 20.4 ± 12.92, n = 99 NSCs. (P) Western blotting analysis of larval brain protein extracts of control (UAS-β-Gal RNAi; UAS-β-Gal RNAi) and hyx knockdown with UAS-Dicer2 (hyx RNAi; UAS-Dicer2 RNAi) driven by actin5C-Gal4 at 48 h ALH. Blots were probed with anti-Hyx antibody and anti-GAPDH antibody. (Q) Fold change of Hyx protein after normalization against GAPDH in brain extracts from control (UAS-β-gal RNAi; UAS-β-gal RNAi) and hyx RNAi with UAS-Dicer2 driven by actin5C-Gal4 at 48 h ALH. Control, 1-fold; hyx RNAi, 0.34 ± 0.07-fold. (R) Western blotting analysis of 48 h ALH larval brain extracts of control (UAS-β-Gal RNAi; UAS-β-Gal RNAi) and hyx knockdown with UAS-Dicer2 (hyx RNAi; UAS-Dicer2 RNAi) under the control of actin5C-Gal4. Blots were probed with anti-Polo antibody, anti-AurA antibody, and anti-GAPDH antibody. (S) Fold change of Polo and AurA protein intensity after normalization against GAPDH in brain extracts from control (UAS-β-gal RNAi; UAS-β-gal RNAi) and hyx RNAi with UAS-Dicer2 driven by actin5C-Gal4 at 48 h ALH. Polo: control, 1-fold; hyx RNAi, 0.25 ± 0.01-fold. AurA: control, 1-fold; hyx RNAi, 0.43 ± 0.00-fold. Two individual repeats were conducted (P and R). Error bars indicate standard deviation in J-S. Arrows indicate the centrosomes in A, D and NSCs in N. Statistical significances were determined by unpaired two-tailed Student t test in B, C, E, K-M, O, Q, and S. ****p < 0.0001 for B, J, M, O, and S; ***p = 0.0004 for tacc in M;**p = 0.0017 for C, **p = 0.0063 for γ-tub in M, and **p = 0.0084 for cnn in M; *p = 0.0391 for E; D, F; **p = 0.0065 for E; **p = 0.0020 for K; ns = 0.6287 for L; ns = 0.1224 for msps in M; **p = 0.0036 for Q; ***p = 0.0002 for S. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. ALH, after larval hatching; Asl, Asterless; AurA, Aurora-A; ChIP-qPCR, chromatin immunoprecipitation coupled with quantitative PCR; Cnn, centrosomin; Hyx, Hyrax; msps, mini spindles; NSC, neural stem cell; RNAi, RNA interference; ROI, region of interest; RT-qPCR, reverse transcription quantitative real-time PCR; TSS, transcription start site; α-tub, α-tubulin; γ-tub, γ-tubulin.

https://doi.org/10.1371/journal.pbio.3001834.g006

To further probe how Hyx regulates centrosome assembly, we examined the ultrastructure of Cnn and γ-tub using super-resolution imaging. Cnn and γ-tub formed “doughnut-like” rings surrounding the centriolar protein Asl, at the centrosomes, in 94.9% and 92.9% of control metaphase cells, respectively (Fig 6F and 6G). Remarkably, Cnn and γ-tub failed to form the ring patterns or formed a ring with reduced inner size at the centrosomes in 51.3% and 53.4% of hyx knockdown mitotic cells, respectively (Fig 6F and 6G). These observations suggest that Hyx is required for the proper recruitment of Cnn and γ-tub at the centrosomes in S2 cells.

Hyx directly regulates the expression of polo and aurA in vitro

Next, we investigated whether Hyx directly regulates the expression of polo and aurA, the 2 key centrosomal proteins. We performed chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (ChIP-qPCR) in S2 cells. After normalizing against “Pre-serum” (1-fold), only a 1.37-fold increase was seen for the negative control. In contrast, 2.94-fold enrichment was observed for orb2 promoter, a positive control. Moreover, we found Hyx binds to the promoter region of polo (new Fig 6I; 2.63-fold and 2.95-fold using 2 pairs of primers). Hyx also binds to the promoter region of aurA (Fig 6I; 2.85-fold), but not numb (Fig 6I; 1.64-fold). Therefore, Hyx binds to the promoter region of both polo and aurA.

We performed the luciferase assay to verify the direct binding of Hyx to the polo promoter. The endogenous Hyx in S2 cells was able to induce the luciferase reporter activity under the control of polo-promoter (poloPro) normalized against Renilla luciferase activity, but not with the vector control (Fig 6J). We attempted to overexpress Hyx in S2 cells to test if it further enhances the luciferase activity under the control of the polo promoter. However, overexpression of Venus-tagged full-length hyx (hyx-FL) resulted in severe cell death (54.3%) detected by active Caspase-3 (S10I and S10J Fig; 11.2% cell death in the control), which precluded us from testing the effect of Hyx overexpression on the transcription of polo in the luciferase assay. Next, we sought to knock down hyx with dsRNA treatment in S2 cells and analyze the relative luciferase activity under the control of the polo promoter. The relative luciferase activity from the ds-hyx treatment group was significantly reduced to 0.5-fold compared with 1-fold from the control group (ds-egfp) (Fig 6K) The relative luciferase activity driven by actin5c promoter induced by ds-hyx treatment and ds-egfp groups was indistinguishable (Fig 6L; 1.0-fold versus 1.2-fold). We conclude that hyx can directly bind to the polo promoter region and promotes its transcription.

The expression of centrosome-related genes depends on Hyx function in larval brains

As Parafibromin/Hyx regulates transcriptional events [78], we wondered whether Hyx was required for the expression of genes that are involved in centrosome assembly. To this end, we sought to perform reverse transcription quantitative real-time PCR (RT-qPCR) to detect differential transcription levels of those genes in larval brains. As both hyx alleles (hyx^HT622 and hyx^w12-46) led to embryonic lethality, we knocked down hyx in the larval brain by RNAi using a ubiquitous driver actin5C-Gal4. However, hyx RNAi under actin5C-Gal4 caused larval lethality after 48 h ALH. Therefore, we performed both RT-qPCR and western blot experiments for hyx RNAi under the control of actin5C-Gal4 at 48 h ALH. hyx mRNA levels were dramatically reduced to 0.23-fold upon hyx knockdown (Fig 6M). The overall Hyx throughout hyx-depleted larval brains was significantly reduced (fluorescence intensity 19.7 ± 8.99 a.u, n = 8 BL), compared with that in control (41.5 ± 13.25, n = 10 BL) (Fig 6N and 6O). Particularly, Hyx levels were dramatically decreased in these hyx-depleted brains (20.4 ± 12.92, n = 99 NSCs), compared with that in control (107.1 ± 29.18, n = 96 NSCs) (Fig 6N and 6O).

Remarkably, mRNA levels of polo and aurA were dramatically decreased to 0.33-fold and 0.24-fold, respectively, following hyx knockdown under actin5C-Gal4 (Fig 6M). Meanwhile, various centrosomal genes were significantly down-regulated upon hyx depletion in the larval brain (Fig 6M; γ-tub, 0.59-fold; cnn, 0.68-fold; tacc, 0.45-fold), but not msps (0.86-fold). Among cell polarity genes, only par6 was significantly decreased to 0.68-fold (S10K Fig), and baz (0.84-fold), pins (1.04-fold), insc (1.07-fold), and numb (1.81-fold) were unaffected. By contrast, aPKC mRNA levels were not significantly affected (S10K Fig, 5.21-fold). The high variation of aPKC mRNA level might be due to unstable aPKC mRNA at the time point of analysis (48 h ALH) when larvae started dying.

Moreover, the western blot result showed that Hyx protein levels were dramatically reduced in hyx RNAi brain under actin5C-Gal4 to 0.34-fold in contrast to 1-fold in control (Fig 6P and 6Q), indicating an efficient hyx knockdown using actin5C-Gal4. Both Polo (0.25-fold) and AurA (0.43-fold) levels normalized against GAPDH levels were dramatically decreased following hyx depletion, compared with 1-fold in control (Fig 6R and 6S).

Taken together, our data suggest that Hyx appears to primarily regulate the expression of polo and aurA in NSCs.

polo and aurA are 2 key downstream targets of Hyx in NSCs

Given that Polo and AurA regulate cell polarity and microtubule functions in NSCs, we sought to investigate whether Polo and AurA are physiologically relevant targets of Hyx in NSCs. We overexpressed polo and aurA in the hyx RNAi knockdown background and found that the ectopic NSC phenotype caused by hyx depletion was significantly suppressed. With the introduction of Venus-polo and aurA into hyx RNAi, the average total NSC number of each brain lobe was significantly reduced to 111.1 ± 10.1 and 114.0 ± 5.1, respectively (Fig 7A and 7B; n = 7 BL and n = 11 BL, respectively) compared with 133.8 ± 4.0 NSCs in hyx RNAi alone (Fig 7A and 7B; n = 5 BL), close to 98.5 ± 2.8 in control larval brains (Fig 7A and 7B; n = 11 BL), Venus-Polo- (99.2 ± 2.4, n = 10 BL) or AurA-overexpressing brains (98.9 ± 3.6, n = 12 BL). Moreover, the average type I NSC number per lineage in hyx RNAi with Venus-polo and aurA overexpression was decreased to 1.7 ± 0.13 (n = 66 NSC lineage) and 2.3 ± 0.32 (n = 27 NSC lineage) (Fig 7C; mean ± SEM), significantly lower than 3.9 ± 0.77 (n = 27 NSC lineage) in hyx RNAi with β-gal RNAi. Likewise, the average type II NSC number per lineage in hyx RNAi with polo and aurA overexpression was significantly dropped to 4.6 ± 0.40 (n = 36 NSC lineage) and 4.5 ± 0.50 (n = 21 NSC lineage), respectively, compared with 7.7 ± 1.4 (n = 19 NSC lineage) in hyx RNAi with β-gal RNAi. Only 1 NSC per lineage was observed in both type I and type II NSC lineages from β-gal RNAi control, Venus-polo overexpression control, and aurA overexpression control.

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Fig 7. Polo and AurA are 2 key downstream targets of Hyx and functional for preventing hyx-depletion mediated NSC overproliferation.

(A) Third instar larval brains from control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), hyx knockdown with UAS control (hyx RNAi; UAS-β-Gal RNAi), hyx knockdown with polo expression (hyx RNAi; UAS-venus-polo) and hyx knockdown with aurA expression (hyx RNAi; UAS-aurA) were labelled with Dpn, Ase, and GFP. RNAi was driven by insc-Gal4. (B) Average NSC number per brain lobe (with SD) for genotypes in A. Control: 98.5 ± 2.4 NSC, n = 11 BL; hyx RNAi; UAS-β-Gal RNAi: 133.8 ± 4.01 NSC, n = 5 BL; venus-polo: 99.2 ± 2.4, n = 10 BL; UAS-aurA: 98.9 ± 3.6, n = 12 BL; hyx RNAi; UAS-venus-polo: 11.1 ± 10.1, n = 7 BL; hyx RNAi; UAS-aurA: 114.0 ± 5.1 NSC, n = 11 BL. (C) Average NSC number per lineage (with SEM) for genotypes in A. Type I: Control, 1.0 ± 0 NSC, n = 20 NSC lineage; hyx RNAi with UAS-β-Gal RNAi, 3.9 ± 0.77 NSC, n = 27 NSC lineage; venus-polo, 1.0 ± 0, n = 34 NSC lineage; UAS-aurA, 1.0 ± 0, n = 36 NSC lineage; hyx RNAi with UAS-venus-polo, 1.7 ± 0.13, n = 66 NSC lineage; hyx RNAi with UAS-aurA, 2.3 ± 0.32 NSC, n = 27 NSC lineage. Type II: Control, 1.0 ± 0 NSC, n = 20 NSC lineage; hyx RNAi with UAS-β-Gal RNAi, 7.7 ± 1.4 NSC, n = 19 NSC lineage; venus-polo, 1.0 ± 0, n = 22 NSC lineage; UAS-aurA, 1.0 ± 0, n = 24 NSC lineage; hyx RNAi with UAS-venus-polo, 4.6 ± 0.40, n = 36 NSC lineage; hyx RNAi with UAS-aurA, 4.5 ± 0.50 NSC, n = 21 NSC lineage. (D) Metaphase NSCs from third instar larval brains of control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), hyx knockdown with UAS control (hyx RNAi; UAS-β-Gal RNAi), hyx knockdown with polo expression (hyx RNAi; UAS-venus-polo) and hyx knockdown with aurA expression (hyx RNAi; UAS-aurA) were stained with aPKC, Mira, PH3, and GFP. RNAi was controlled by insc-Gal4. (E) Percentage of NSC with indicated levels of aPKC crescent for genotypes in D. Control (n = 58 NSC): Strong, 94.9%; Weak, 5.2%; No, 0; hyx knockdown with UAS control (n = 55 NSC): Strong, 0; Weak, 26.6%; No, 73.4%; venus-polo (n = 25 NSC): Strong, 95.0%; Weak, 5.0%; No, 0; UAS-aurA (n = 24 NSC): Strong, 96.5%; Weak, 3.6%; No, 0; hyx knockdown with polo expression (n = 59 NSC): Strong, 4.1%; Weak, 48.9%; No, 47.1%; hyx knockdown with aurA expression (n = 51 NSC): Strong, 6.5%; Weak, 54.7%; No, 38.8%. (F) Percentage of NSC with indicated levels of Mira crescent for genotypes in D. Control (n = 58 NSC): Strong, 94.9%; Weak, 5.2%; No, 0; hyx knockdown with UAS control (n = 55 NSC): Strong, 0; Weak, 29.8%; No, 70.3%; venus-polo (n = 25 NSC): Strong, 96.7%; Weak, 3.4%; No, 0; UAS-aurA (n = 24 NSC): Strong, 91.5%; Weak, 8.6%; No, 0; hyx knockdown with polo expression (n = 59 NSC): Strong, 1.4%; Weak, 57.0%; No, 41.7%; hyx knockdown with aurA expression (n = 51 NSC): Strong, 6.5%; Weak, 51.5%; No, 42.0%. (G) Metaphase NSCs of control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), hyx knockdown with UAS control (hyx RNAi; UAS-β-Gal RNAi), hyx knockdown with polo expression (hyx RNAi; UAS-venus-polo) and hyx knockdown with aurA expression (hyx RNAi; UAS-aurA) under the control of insc-Gal4 were labelled for α-tub, GFP, and DNA. (H) Ratio of spindle length to NSC diameter (with SD) for genotypes in G. Control: 0.90 ± 0.05, n = 18; hyx knockdown with UAS control: 0.82 ± 0.06, n = 23; hyx knockdown with polo expression: 0.92 ± 0.06, n = 22; hyx knockdown with aurA expression: 0.91 ± 0.06, n = 27. (I) A working model. One-way ANOVA with multiple comparison was performed in B, C and H. ****p < 0.0001 in B, C and H; ns > 0.9999 in B; ns = 0.9647 and 0.9874 in H. For type I group from C: **p = 0.0035, *p = 0.0350, and ns = 0.3465. For type II group from C: ***p = 0.0005, ***p = 0.0003, and **p = 0.0012.Two-way ANOVA with multiple comparison was performed in E and F. ****p < 0.0001 in E and F; for “Weak aPKC crescent" in E: **p = 0.0028; ***p = 0.0002. For “No aPKC crescent" in E: ***p = 0.0003. For “Weak Mira crescent" in F: **p = 0.0029, **p = 0.0010, and **p = 0.0089. For “No Mira crescent" in F: ***p = 0.0006, and ***p = 0.0007. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. aPKC, atypical PKC; AurA, Aurora-A; Hyx, Hyrax; Mira, Miranda; NSC, neural stem cell; RNAi, RNA interference; α-tub, α-tubulin.

https://doi.org/10.1371/journal.pbio.3001834.g007

Importantly, cell polarity defects caused by hyx depletion were also significantly suppressed by overexpression of polo and aurA in NSCs. The majority (73.4%) of hyx RNAi metaphase NSCs had lost aPKC polarity and the remaining 26.6% of NSCs had a weak aPKC crescent (Fig 7D and 7E; n = 55). Remarkably, 4.1% of hyx RNAi NSCs with Venus-polo overexpression formed a strong aPKC crescent and 48.9% showed a weak aPKC crescent (Fig 7D and 7E; n = 59 NSC). Similarly, a strong Mira crescent was observed in 1.4% of hyx RNAi NSCs with Venus-polo overexpression and 57.0% formed a weak Mira crescent (Fig 7D and 7F; n = 59), compared with 29.8% of NSCs with a weak Mira crescent in hyx knockdown alone (Fig 7D and 7F; n = 55). Likewise, aurA overexpression can dramatically restore the asymmetric localization of both aPKC and Mira in hyx RNAi NSCs. Upon aurA overexpression, a strong aPKC crescent was seen in 6.5% of hyx RNAi NSCs and a weak aPKC crescent was formed in 54.7% of NSCs (Fig 7D and 7E; n = 51 NSC). Similarly, upon aurA overexpression, 6.5% of hyx RNAi NSCs formed a strong Mira crescent and 51.5% showed a weak Mira crescent (Fig 7D and 7F; n = 51 NSC). These suppressions were partial because the vast majority of metaphase NSCs from control (n = 58), Venus-Polo- (n = 25), or AurA-overexpression (n = 24) assembled strong aPKC and Mira crescent on the cortex (Fig 7D). Moreover, the shorter spindle phenotype in hyx RNAi was also significantly suppressed by polo and aurA overexpression (Fig 7G and 7H; control, 0.90 ± 0.05, n = 18 NSCs; 0.82 ± 0.06, n = 23 NSCs; 0.92 ± 0.06, n = 22 NSCs; 0.91 ± 0.06, n = 27 NSCs). These results support our conclusion that both Polo and AurA are physiologically relevant targets of Hyx in NSCs. Therefore, down-regulated polo and aurA expression likely accounts for various defects, such as loss of asymmetry of apical and basal proteins and centrosome/microtubule abnormalities, observed in hyx-depleted NSCs. Polo can be directly activated by AurA in Drosophila mitotic NSCs [79]. Our data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly (Fig 7I).

Fly stocks and genetics

Fly stocks and genetic crosses were reared at 25°C unless otherwise stated. Fly stocks were kept in vials or bottles containing standard fly food (0.8% Drosophila agar, 5.8% Cornmeal, 5.1% Dextrose, and 2.4% Brewer’s yeast). The following fly strains were used in this study: insc-Gal4 (BDSC#8751; 1407-Gal4), insc-Gal4, UAS-Dicer2 with and without UAS-CD8-GFP, Jupiter-GFP (G147), UAS-hyx, UAS-HRPT2 [51]. The type II NSC driver: w; UAS-Dicer 2, wor-Gal4, ase-Gal80/CyO; UAS-mCD8-GFP/TM3, Ser [86]; hyx RNAi hyx^HT622, UAS-venus-polo [28].

The following stocks were obtained from Bloomington Drosophila Stock Center (BDSC): UAS-Gal RNAi (BDSC#50680; this stock is often used as a control UAS element to balance the total number of UAS elements), ctr9^12P023 (BDSC#59389), rtf1 RNAi (BDSC#36586), rtf1 RNAi (BDSC#34850) [87], rtf1 RNAi (BDSC#31718), UAS-aurA (BDSC#8376), actin5C-Gal4 (BDSC#25374).

The following stocks were obtained from Vienna Drosophila Resource Center (VDRC): hyx RNAi (28318), hyx RNAi (103555), atms RNAi (108826) [87], atu RNAi (17490) [88], atu RNAi (106074), ctr9 RNAi (108874), ctr9 RNAi [89], rtf1 RNAi (27341) [88], rtf1 RNAi (110392).

All experiments were carried out at 25°C, except for RNAi knockdown or overexpression experiments that were performed at 29°C.

Immunohistochemistry

Third instar Drosophila larvae were dissected in PBS, and larval brains were fixed in 4% EM-grade formaldehyde in PBT (PBS + 0.3% Triton-100) for 22 min. The samples were processed for immunostaining as previously described [77]. For α-tubulin immunohistochemistry, larvae were dissected in Shield and Sang M3 medium (Sigma-Aldrich), supplemented with 10% FBS, followed by fixation in 10% formaldehyde in Testis buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA (pH 6.8)), supplemented with 0.01% Triton X-100. The fixed brains were washed once in PBS and twice in 0.1% Triton X-100 in PBS. Images were taken with an AxioCam HR camera (with 1.5× to 8× of digital zoom) of a LSM710 confocal microscope system (Axio Observer Z1; ZEISS), using a Plan-Apochromat 40×/1.3 NA oil differential interference contrast objective. The brightness and contrast of the images obtained were adjusted using Adobe Photoshop or Fiji (imageJ).

The primary antibodies used were the following: rabbit affinity-purified anti-Hyx/Cdc73 (1:1,000; J. T. Lis); guinea pig anti-Dpn (1:1,000), mouse anti-Mira (1:50; F. Matsuzaki), rabbit anti-Mira (1:500, W. Chia), anti-Insc (1:1,000, X. Yang), rabbit anti-aPKCζ C20 (1:100; Santa Cruz Biotechnologies, Dallas, TX), guinea pig anti-Numb (1:1,000; J Skeath), rabbit anti-GFP (1:3,000; F. Yu), mouse anti-GFP (1:5,000; F. Yu), rabbit anti-Asense (1:1,000; YN Jan), guinea pig anti-Asl (1:200, C. Gonzalez), rabbit anti-Sas-4 (1:100, J. Raff), mouse anti-α-tubulin (1:200, Sigma, Cat#: T6199), mouse anti-γ-tubulin (1:200, Sigma, Cat#: T5326), rabbit anti-Cnn (1:5,000, E. Schejter and T. Megraw), rabbit anti-Msps (1:500), rabbit anti-Msps (1:1,000, J. Raff), rabbit anti-PH3 (1:200, Sigma, Cat#: 06–570), rabbit anti-DTACC (1:200), rabbit anti-Ana2 [24], α-tubulin (1:200, Sigma, Cat#: T6199), rabbit anti-AurA (1:200, J. Raff), rat anti-CD8 (1:250, Caltag Laboratories), mouse anti-Polo (1:30, C. Sunkel), rabbit anti-Cleaved Caspase-3 (Asp175) (1:100, Cell Signaling, Cat#: 9664), rabbit anti-Phospho Drosophila Akt (Ser505) (1:100, Cell Signaling, Cat#: 4054), rabbit anti-Stg/Cdc25 (1:500, Eric F. Wieschaus), rabbit anti-Mts (1:50). The secondary antibodies used were conjugated with Alexa Fluor 488, 555, or 647 (Jackson Laboratory).

Spinning disc super-resolution imaging

Super-resolution Spinning Disc Confocal-Structured Illumination Microscopy (SDC-SIM) was performed on a spinning disk system (Gataca Systems) based on an inverted microscope (Nikon Ti2-E; Nikon) equipped with a confocal spinning head (CSU-W; Yokogawa), a Plan-Apo objective (100×1.45-NA), and a back-illuminated sCMOS camera (Prime95B; Teledyne Photometrics). A super-resolution module (Live-SR; GATACA Systems) based on structured illumination with optical reassignment technique and online processing leading to a 2-time resolution improvement [90] is included. The maximum resolution is 128 nm with a pixel size of 64 nm in super-resolution mode. Excitation light at 488 nm/150 mW (Vortran) (for GFP), 561 nm/100 mW (Coherent) (for mCherry/mRFP/tagRFP) and 639 nm/150 mW (Vortran) (for iRFP) was provided by a laser combiner (iLAS system; GATACA Systems), and all image acquisition and processing were controlled by the MetaMorph (Molecular Device) software. Images were further processed with imageJ.

Generation of guinea pig anti-Hyx antibodies

The cDNA region encoding the N-terminal 1–176 amino acid residues of Hyx/Cdc73 was amplified by PCR with the oligos: 5′-TCCGAATTCATGGCAGATCCGCTCA GC-3′ and 5′-ATGCGGCCGCCTACGTCTCGGACAGCGACTT-3′. The PCR products were cloned into the pMAL-c2x (Addgene # 75286) vector. The fusion protein MBP-Hyx 1–176 was purified and injected into guinea pigs, and the antibodies generated were purified by GenScript (Hong Kong).

Clonal analysis

MARCM clones were generated as previously described [54]. Briefly, larvae were heat shocked at 37°C for 90 min at 24 h ALH and 10 to 16 h after the first heat shock. Larvae were further aged for 3 d at 25°C, and larval brains were dissected and processed for immunohistochemistry. To generate type II NSC clones, UAS lines were crossed to the type II driver (worniu (wor)-Gal4, ase-Gal80^ts; UAS-CD8-GFP) at 25°C and shifted to 29°C at 24 h ALH. Wandering third instar larvae were dissected after incubation for 3 or 4 d at 29°C. Z-stacks were acquired and NSC number per clone/lineage was manually counted. Percentage of ectopic NSCs refers to the percentage of NSC clones or lineages with ectopic NSCs out of total number of clones/lineages scored in this study. All clones in the larval brains scored in this study were NSC clones.

Time-lapse recording

The time-lapse recording was performed as described [44]. The whole-mount brain expressing G147-GFP was used to analyze the asymmetric cell division of NSCs. The brain was dissected and loaded into a Lab-Tek chambered coverglass (Thermo Fisher Scientific) filled with dissecting medium that is supplemented with 2.5% methyl cellulose (Sigma-Aldrich). The time-lapse images of NSC divisions were acquired every 30 s on a confocal microscope (LSM 710; ZEISS). The video was processed with ImageJ and displayed at 15 frames per second.

Microtubule regrowth assay

The microtubule regrowth assay was performed as described previously [44]. Third instar larval brains were dissected in Shield and Sang M3 insect medium (Sigma-Aldrich) supplemented with 10% FBS, and microtubules were depolymerized by incubating the larval brains on ice for 40 min. The brains were allowed to recover at 25°C for various time periods to facilitate microtubule regrowth. The brains were immediately fixed in 10% formaldehyde in testis buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA (pH 6.8)) supplemented with 0.01% Triton X-100. The fixed brains were washed once in PBS and twice in 0.1% Triton X-100 in PBS, following which they were processed for immunohistochemistry. The mean immunofluorescence intensity of α-tub detected on astral microtubules proximal to MTOC at interphase and spindle microtubules at metaphase were quantified on selected regions of the same size with ImageJ.

S2 cell culture, transfection, and quantitative RT-PCR

Chromatin immunoprecipitation

ChIP was performed according to the manufacturer’s protocol (Cell Signaling, #9005). Sonicated lysates were used for ChIP with antibodies against Hyx (final bleed, J. T. Lis) and preimmune serum as a control. Immunoprecipitated DNA was analyzed by quantitative real-time PCR using specific primers to the potential promoter region of various centrosomal genes and negative control—the intergenic sequence at 5 kb downstream of numb genome as well as the positive control targeting the potential orb2 promoter region:

polo Pro forward: 5′-TACCAGAAAGTGTGCGATAGCC-3′;

polo Pro reverse: 5′-GAAACGGAGATCAGATCCACAC-3′;

polo Pro forward: 5′-CCTGCAATTACAAGGTGGCA-3′;

polo Pro reverse: 5′-ACTAAACAGTCAACGGTCAACT-3′;

aurA Pro forward: 5′-TCGGCATCATATCATAAACGAC-3′;

aurA Pro reverse: 5′-TTATCGGGCATCTCTGAACA-3′;

numb Pro forward: 5′-CAGCCCAACAAGCCAATAAA-3′;

numb Pro reverse: 5′-GGGCGTGAGTAAATTGTCGT-3′;

negative control forward: 5′-TCCTTGGTCCTAACGTGGTC-3′;

negative control reverse: 5′-AAGTATTTGCCCCAGCTTGA-3′;

orb2 Pro forward: 5′-CTCCACAACGATTCCGATTT-3′;

orb2 Pro reverse: 5′-CCGCACCAACACTTTCTACA-3′.

Luciferase assay

Luciferase assay was performed according to the manufacturer’s protocol (Dual-Glo Luciferase Assay System (E2920), Promega). Drosophila S2 cells were cotransfected with 0.1 μg of polo promoter-luciferase reporter (poloPro-luc) and 0.1 μg of Venus-tagged hyx-FL. Luciferase reporter is made on the pGL3 basic reporter construct (Promega), and 643 bp sequence before the transcriptional start site of polo genome was cloned into the pGL3-basic vector bone. The Venus-tagged hyx is made by cloning full-length wild-type hyx into pAVW (Drosophila Genomics Resource Center [DGRC]). For negative control, pGL3-basic and empty pAVW was cotransfected into S2 cells. For normalizing transfection efficiency, 0.1 μg of plasmid encoding the Renilla gene was transfected. S2 cells were treated with ds-egfp and ds-hyx for 24 h before transfection with 0.1 μg of pGL3-poloPro and 0.1 μg of pRL-SV40 plasmids and cells were harvested 48 h after transfection. For the internal control group, 0.1 μg of actin5c promoter containing pGL3 (actin5c-luc) was cotransfected with 0.1 μg of pRL-SV40 plasmid into S2 cells.

Western blotting

Embryos were treated with 50% bleach for 2 min to dissolve chorion. After that, embryos or larval brains were homogenized in RIPA buffer (50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate,0.1% SDS), and western blotting was carried out according to standard procedures.

Quantification and statistical analysis

Drosophila larval brains from various genotypes were placed dorsal side up on confocal slides. The confocal z-stacks were taken from the surface to the deep layers of the larval brains. For each genotype, at least 10 NSCs were imaged and ImageJ or Zen software was used for quantifications.

The localization of polarity proteins was scored by 3 categories: “Strong crescent,” “Weak crescent,” and “No crescent.”

Statistical analysis was essentially performed using GraphPad Prism 9. Unpaired two-tail t tests were used for comparison of 2 sample groups, and one-way ANOVA or two-way ANOVA followed by Sidak’s multiple comparisons test was used for comparison of more than 2 sample groups. All data are shown as the mean ± SD. Statistically nonsignificant (ns) denotes p > 0.05, * denotes p <0.05, ** denotes p <0.01, *** denotes p <0.001, and **** denotes p < 0.0001. All experiments were performed with a minimum of 2 repeats. In general, n refers to the number of NSCs counted unless otherwise indicated.