ABSTRACT
Neuroblasts in flies divide asymmetrically by establishing polarity, distributing cell fate determinants asymmetrically, and positioning their spindle for cell division. The apical complex contains aPKC, Bazooka (Par3), and Par6, and its activity depends on L(2)gl. We show that Ankle2 interacts with L(2)gl and affects aPKC. Reducing Ankle2 levels disrupts ER and nuclear envelope morphology, releasing the kinase Ballchen/VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen/VRK1 or l(2)gl suppresses the loss of Ankle2 and restores viability and brain size. The Zika virus NS4A protein interacts with Drosophila Ankle2 and VRK1 in dividing neuroblasts. Human mutational studies implicate this neural cell division pathway in microcephaly and motor neuron disease. In summary, NS4A, ANKLE2, VRK1 and LLGL1 define a novel pathway that impinges on asymmetric determinants of neural stem cell division.
INTRODUCTION
Proper development of the human brain requires an exquisitely coordinated series of steps and is disrupted in disorders associated with congenital microcephaly. Congenital microcephaly is characterized by reduced brain size (using occipital frontal circumference, OFC, as a surrogate measure) more than two standard deviations below the mean (Z-score < −2) at birth. It is associated with neurodevelopmental disorders, such as developmental delay and intellectual disability (DD/ID) and can be caused by external exposures to toxins, in utero infections, or gene mutations. Pathogenic gene variants for microcephaly have been identified through targeted genetic testing, genomic copy number studies, and exome sequencing (ES) (Brunetti-Pierri et al., 2008; Dumas et al., 2012; Lupski, 2015; Shinawi et al., 2010; Shaheen et al., 2018), elucidating about 18 primary microcephaly loci. Many syndromes significantly overlap with classic microcephaly phenotypes, and together, these disorders can be caused by defects in a wide variety of biological processes, including centriole biogenesis, DNA replication, DNA repair, cell cycle and cytokinesis, genome stability, as well as multiple cell signaling pathways (Jayaraman, Bae and Walsh, 2018).
A forward, mosaic screen for neurodevelopmental and neurodegenerative phenotypes associated with lethal mutations on the X-chromosome in Drosophila identified 165 loci, many with corresponding human genetic disease trait phenotypes (Yamamoto et al., 2014). Among them, a mutation in Ankle2 (Ankryin repeat and LEM domain containing 2) causes loss of Peripheral Nervous System (PNS) organs in adult mutant clones and severely reduced brain size in hemizygous third instar larvae. To identify patients with pathogenic variants in ANKLE2, we surveyed the exome database of the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) (Bamshad et al., 2012; Posey et al., 2019) and identified compound heterozygous mutations in ANKLE2 in two siblings. Both infants exhibited severe microcephaly (Z-score = −9), and the surviving patient displayed cognitive and neurological deficits alongside extensive intellectual and developmental disabilities. Previously, we showed that mutations in Ankle2 lead to cell loss of neuroblasts and affected neuroblast division in the developing third instar larval brain. Remarkably, expression of the wild type human ANKLE2 in flies rescued the observed mutant phenotypes (Yamamoto et al., 2014). Here we explore the molecular pathways and proteins that are affected by Ankle2 loss.
ANKLE2 belongs to a family of proteins containing LEM (LAP2, Emerin, MAN1) domains that typically associate with the inner nuclear membrane (Lin et al., 2000; Barton, Soshnev and Geyer, 2015). Conventional LEM proteins have been shown to interact with BAF (Barrier to Autointegration Factor), which binds to both DNA and the nuclear lamina (Segura-Totten et al., 2002) to organize nuclear and chromatin structure. However, the LEM domain in Drosophila and C. elegans Ankle2 is not obviously conserved (Marchler-Bauer et al., 2017). Studies in C. elegans indicate that a homolog of ANKLE2 regulates nuclear envelope morphology and functions in mitosis to promote reassembly of the nuclear envelope upon mitotic exit (Asencio et al., 2012; Snyers et al., 2018). During this process, ANKLE2 modulates the activities of VRK1 (Vaccina Related Kinase 1) and PP2A (Protein Phosphatase 2A) (Asencio et al., 2012). However, all experiments in worms were performed at the embryonic two-cell stage, and no other phenotypes were reported except early lethality. Whilst mutations in ANKLE2 have been associated with severe microcephaly (OFC z-sore = −2.5 to −9), human VRK1 pathogenic variant alleles can cause a neurological disease trait consisting of complex motor and sensory axonal neuropathy and microcephaly (Gonzaga-Jauregui et al., 2013).
Mutations in both Ankle2 and the fly homologue of VRK1, ballchen, cause a loss of neuroblasts in 3rd instar larval brains in Drosophila (Yamamoto et al., 2014; Yakulov et al., 2014). Neuroblasts (NBs) divide asymmetrically and are often used as a model to investigate stem cell biology (Homem and Knoblich, 2012) and asymmetric cell division (Gallaud et al., 2017). Most NBs in the larval central brain give rise to another NB and a smaller ganglion mother cell (GMC), which then divides once again to produce neurons or glia. Proper NB maintenance and regulation is essential for precise development of the adult nervous system, and misregulation of NB number or function can lead to defects in brain size (Wang et al., 2009; Gateff and Schneiderman, 1974).
Congenital Zika infection in humans during pregnancy has been associated with severe microcephaly that can be as dramatic as certain genetic forms of microcephaly including phenotypes associated with bi-allelic mutations in MCPH16/ANKLE2 (Moore et al., 2017; Yamamoto et al., 2014). Recently, we showed that a Zika protein, NS4A, physically interacts with ANKLE2 in human cells. Expression of NS4A in larval brains causes microcephaly, induces apoptosis, and reduces proliferation. Importantly, expression of human ANKLE2 in flies that express NS4A suppresses the associated phenotypes, demonstrating that NS4A interacts with the ANKLE2 protein and inhibits its function (Shah et al., 2018). Interestingly, the Zika virus crosses the blood brain barrier and targets radial glial cells, the neural progenitors in the vertebrate cortex (Devhare et al., 2017; Tang et al., 2016).
Here, we show that Ankle2 is localized to the endoplasmic reticulum and nuclear envelope, like NS4A (Shah et al., 2018), and genetically interacts with ball/VRK1 to regulate brain size in flies. An allelic series at the ANKLE2 and VRK1 loci shows that perturbation of this pathway results in neurological disease including microcephaly. Our data indicate that the Ankle2-Ball/VRK1 pathway is required for proper localization of asymmetric proteins and spindle alignment during NB cell division by affecting two proteins, aPKC and L(2)gl, that play critical roles in the asymmetric segregation of cell fate determinants. In addition, NS4A expression in neuroblasts mimics phenotypes seen in Ankle2 mutants, and NS4A induced microcephaly is suppressed by removing a single copy of ball/VRK1. Human genomics variant data and disease trait correlations extend this asymmetric cell division pathway from proteins identified in flies and reveal insights into neurological disease. In summary, NS4A hijacks the Ankle2-Ball/VRK1 pathway, which regulates progenitor stem cell asymmetric division during brain development and defines a novel human microcephaly pathway.
RESULTS
Human ANKLE2 variants cause microcephaly
We previously reported that compound heterozygous variants in ANKLE2 are associated with microcephaly (Z-score = −9) (MCPH16, MIM#616681) in two affected siblings (Yamamoto et al., 2014). Here, we report two additional probands carrying unique variants in ANKLE2 identified in Seattle (LR17-511 and LR18-033; Figure 1 and S1, Table S1). Brain MRIs of an age matched control (Figure 1A) and a proband with microcephaly from family LR17-511 document one of the more severe cases of microcephaly (Z-score = −8) (Figure 1B). To investigate potential genotype-phenotype correlations, we explored the spectrum of reported neurological disease trait manifestations associated with ANKLE2 present in the Baylor Genetics (BG) Laboratories databases. These contain clinical exome sequencing (ES) of patients with presumed genetic disorders. We screened for rare biallelic variants, predicted damaging, in ANKLE2 that fulfill Mendelian expectations for a recessive disease trait. Three families were found to fulfill these criteria in probands with neurologically associated phenotypes (Figure 1D and S1, Table S2). These cases suggest that a diverse set of variants in ANKLE2 may be associated with a spectrum of neurologic disease (Figure 1) and reveal either sporadic disease, apparent vertical transmission, and in some cases, consanguineous parentage (Yamamoto et al., 2014; Shaheen et al., 2018); (Figure S1). The identified mutations are missense, nonsense, or splicing variants that lead to premature stop codons; all subjects have biallelic variants, either compound heterozygous or homozygous alleles (Figure 1 and S1). Probands exhibit congenital microcephaly (Figure 1D), but some also present with severe brain MRI abnormalities and skin pigmentation abnormalities (Figure 1D). These aggregate data demonstrate that mutations in ANKLE2 cause autosomal recessive microcephaly.
Null alleles of Ankle2 are associated with reduced brain size in flies
Given the human genetic implications noted above, we used Drosophila to elucidate molecular mechanisms underlying ANKLE2 associated microcephaly. The mutation originally identified in flies, Ankle2A (L326A), causes reduced brain size in third instar larvae and leads to pupal lethality at temperatures ≥ 22°C (Figure S2). It results in decreased neuroblast number, reduced cell divisions, and a high incidence of apoptotic cell death (Yamamoto et al., 2014). To create a severe loss of function allele for Ankle2, we integrated a CRIMIC construct containing attP-FRT-SA-3XSTOP-polyA-3xP3-EGFP-FRT-attP sequences using CRISPR-Cas9 in the fifth intron shared by all isoforms (Figure 2A; pM14; (Lee et al., 2018)). The polyA sequences arrest transcription, leading to a truncated transcript that likely corresponds to a null allele (Ankle2CRIMIC, Figure 2A). These animals die as 3rd instar larvae (Figure 2B), are smaller than wild type and Ankle2A animals, and show severely reduced brain volume (Figure 2E versus I, and M) with complete disruption of the optic lobe (Figure 2I).
To determine whether Ankle2 is expressed in the brain, we used the CRIMIC allele to introduce an artificial exon that contains SA-GFP-SD in frame which produces a tagged fusion protein (Ankle2IGFP, Figure 2A-B) (Lee et al., 2018). We readily detect Ankle2IGFP protein in brains of heterozygous animals (Figure S3). However, homozygous animals are lethal and exhibit very small brains, indicating that integration of this exon disrupts protein function. Based on complementation tests, the strength of the allelic series is Ankle2A < Ankle2CRIMIC= Ankle2IGFP (Figure 2B and C). We therefore used recombineering (Venken et al., 2006) to add a C-terminal GFP tag to Ankle2 in a BAC (CH321-85N12; referred to as Ankle2-GFPR, Figure 2A and D) (Venken et al., 2009). When this P[acman] clone was introduced in all three Ankle2 mutant backgrounds, Ankle2-GFPR rescued brain phenotypes and lethality of these alleles (Figure 2B, G, and J). Hence, the chromosomes carrying the three Ankle2 alleles do not carry second site mutations that affect brain size or viability and the tagged protein is likely to reflect the endogenous Ankle2 protein distribution.
The human reference ANKLE2 gene rescues lethality and small brain phenotypes of Ankle2A animals when expressed ubiquitously (da-GAL4 >UAS-hANKLE2, Figure 2H, M). To determine whether the microcephaly associated mutations in human ANKLE2 are loss of function alleles, we next expressed ANKLE2 p.L573V, ANKLE2 p.Q782*, ANKLE2 p.A109P, ANKLE2 p.G201W, in Ankle2A mutant animals. The p.Q782*, p.A109P, and p. G201W variants failed to rescue lethality or reduced brain sizes (Figure 2L-M) consistent with them being severe loss-of-function variant alleles. However, p.L573V restored both viability and brain size (Figure 2K, M) in some Ankle2A animals (Figure 2M), indicating that this variant is a mild hypomorphic allele.
Ankle2 localizes to the ER and nuclear envelope and is required for their integrity
The tagged genomic rescue construct, Ankle2-GFPR (Figure 3A-B), as well as the endogenously tagged Ankle2IGFP (Figure S3) show that Ankle2 is expressed in most cells of the third instar larval brain. The protein appears to be localized to the cytoplasm of all cells including neuroblasts (arrows in Figure 3A-B). However, in a subset of cells, the protein is clearly enriched at the nuclear envelope (arrowhead). To determine the dynamics of Ankle2 protein localization, we performed live imaging. As shown in Movies S1 and S2, the protein is recruited to the nuclear envelope at the initiation of mitosis and remains associated with the nuclear envelope until briefly after cytokinesis.
To determine precisely where Ankle2 is localized, we performed live imaging of brains from animals carrying Ankle2-GFPR and a transgene that labels the ER: da-Gal4>UAS-Sec61β-tdTomato (Summerville et al., 2016). In neuroblasts (large cell in Figure 3C-E), the Ankle2 protein fully colocalizes with Sec61β at the nuclear envelope as well as the ER. In the surrounding neurons (small cells), much of the cytoplasm is colabeled. We also counter-stained fixed samples with Calnexin 99a, another ER marker (Riedel et al., 2016). Again, Ankle2 localizes to the nuclear envelope and the ER, but in fixed samples, the ER structure is less obvious than in live imaging (Figure 3G-I).
To determine if Ankle2 is required for proper ER structure, we performed live imaging of Ankle2A mutant neuroblasts expressing Sec61β-tdTomato (Figure 3F). When compared to wild type (Figure 3E), Ankle2A mutants display highly aberrant Sec61β localization in many NBs (25°C). In addition, we stained fixed Ankle2A mutant neuroblasts with Calnexin 99a and found that Ankle2A mutants also display irregular Calnexin 99a localization (Figure 3J versus 3I), suggesting that even a partial loss of Ankle2 disrupts ER and possibly nuclear envelope structure. Indeed, the morphology of the nuclear envelope is aberrant and convoluted in some Ankle2A mutant cells when stained with Lamin DmO (Riemer et al., 1995), a nuclear envelope marker (compare Figure 3N with Figure 3K-M). Hence, Ankle2 is required for proper ER and nuclear envelope morphology.
Ankle2 mutations affect the asymmetric localization of neuroblast determinants
Due to the reduced cell proliferation and reduced neuroblast number in Ankle2A third instar brains (Yamamoto et al., 2014), we sought to explore neuroblast division in more detail. Neuroblast polarity during division relies on the function of the highly conserved apically localized Par complex, which consists of Bazooka (Par3) (Schober et al., 1999), Par6 (Petronczki and Knoblich, 2001), and atypical Protein Kinase C (aPKC) (Rolls et al., 2003). Once activated, the Par complex is responsible for restricting Miranda (Mira) and other cell fate determinants to the basal domain of neuroblasts. After division in most neuroblasts, the basal domain will become the ganglion mother cell, which divides again to produce neurons or glia (Betschinger et al., 2003; Atwood and Prehoda, 2009). Several proteins have been implicated in regulating the Par complex (Chabu and Doe, 2009; Andersen et al., 2012; Bonaccorsi et al., 2007; Atwood et al., 2007), including those associated with cell cycle regulation (Chabu and Doe, 2008; Lee et al., 2006; Wang et al., 2007; Wang et al., 2006).
Staining of third instar Ankle2A brains with anti-Bazooka, Par6, aPKC, and Mira revealed severe localization defects of these proteins in greater than 40% of metaphase neuroblasts during asymmetric division (Figure 4A-L, quantified in Figure 4M-P) in both Ankle2A and trans-heterozygous animals (Ankle2A/Ankle2CRIMIC). These defects are rescued by the genomic construct (Figure 2D), Ankle2-GFPR (Figure 4D, H, L, M-P). Finally, we performed live imaging of 3rd instar larval brains of wild type (Movie S3) and Ankle2A mutants labeled with Mira-RFP and Histone-GFP (Movies S4-S6). As shown in Movies S4-S6, neuroblasts exhibit abnormal Mira localization as well as some instances of failed division including DNA segregation defects, chromatin bridges, and cytokinesis defects (Movies S4-S5).
For proper neuroblast division to occur, cells must not only asymmetrically localize Par complex members and cell fate determinants, they must also align the mitotic spindle so that divisions segregate cell fate determinants to the proper daughter cell (Cabernard and Doe, 2009). In wild type neuroblasts, the mitotic spindle is aligned parallel to the polarity axis (Figure 4Q, T). In some Ankle2A mutant neuroblasts, we noted that spindle alignment appeared disrupted. To quantify these defects, we measured the axis of division using DNA and Centrosomin (CNN) (Lucas and Raff, 2007) to highlight centrosome placement relative to the localization of cell polarity proteins aPKC and Mira (Figure 4Q-U). Surprisingly, we found that nearly 40% of Ankle2A mutant neuroblasts contained supernumerary centrosomes (Figure 4S). In the remaining 60% of Ankle2A mutant metaphase neuroblasts with obvious aPKC/Mira localization, we found varying degrees of mitotic spindle alignment defects (compare Figure 4Q-R and 4T-U), showing that Ankle2 is also required for proper spindle alignment in neuroblast division. Together, these results show that Ankle2 plays a prominent role in asymmetric protein localization, spindle alignment, and cell division of neuroblasts.
Ankle2 interacts with VRK1/Ballchen
The C. elegans homologue of Ankle2, lem4L, was previously shown to physically and genetically interact with VRK1, the homologue of Ballchen (Ball) in flies (Asencio et al., 2012). Lem4L and VRK1 in worms localize to the nuclear envelope of the 2-cell stage embryo (Asencio et al., 2012). In contrast, Ball is localized to the nucleus in fly neuroblasts (Yakulov et al., 2014). Interestingly, human VRK1 pathogenic variants cause reduced brain size and microcephaly as well as axonal neuropathy in affected patients (Gonzaga-Jauregui et al., 2013; Renbaum et al., 2009). Hence, to characterize the relationship between Ankle2 and Ball/VRK1, we analyzed the expression and localization of Ball and Ankle2 during neuroblast cell division (Figure 5A-D). During interphase, Ankle2 and Ball do not colocalize as Ankle2 is in the cytoplasm and ER whereas Ball is in the nucleus (Figure 5A). During the mitotic prophase, Ankle2 accumulates at the nuclear envelope but the proteins do not seem to colocalize (Figure 5B). However, upon fragmentation of the nuclear envelope during metaphase, Ball is briefly present throughout the cytoplasm (Figure 5C). Yet, during telophase, Ball is quickly recruited back to the nucleus and briefly enriched at the nuclear envelope (Figure 5D; Movie S7). Interestingly, the spatial restriction of Ball in Ankle2A mutants during interphase is abolished in many neuroblasts as Ball localizes throughout the cell, a phenotype that is not observed in wild type brains (Figure 5E-F). In summary, Ankle2 is required for proper nuclear localization of Ball in Drosophila.
To determine whether ANKLE2 regulates Ball/VRK1 subcellular localization in human cells, we assayed VRK1 localization in human fibroblasts. In reference human primary fibroblasts (parental variant p.L573V/+), VRK1 is localized to the nucleus (Figure 5G). However, fibroblasts from microcephaly patients carrying compound heterozygous variants in ANKLE2 (p.L573V/p.Q782* and p.V229G/p.V229G) display significantly reduced VRK1 intensity in the nucleus (Figure 5H, quantified in Figure 5I) and increased cytoplasmic staining in non-dividing cells (arrows in Figure 5H) with no significant change in overall VRK1 intensity (Figure S4). These data argue for a conserved role between fruit flies and human for ANKLE2 in restricting VRK1 to the nucleus.
Given that Ankle2 is required to maintain Ball/VRK1 in the nucleus during interphase, it is possible that Ball/VRK1 is ectopically active in the cytoplasm of Ankle2A mutants and inhibits or promotes phosphorylation of proteins not normally encountered in the biological homeostatic state. Reducing the level of Ball/VRK1 may therefore alleviate the phenotype associated with the reduction in Ankle2 protein. Indeed, we observe evidence for strong dominant interactions between Ankle2A and ball (multiple alleles). Ankle2A animals are pupal lethal and have reduced brain volumes (compare Figure 5J to 5K). However, removal of one copy of ball, akin to a heterozygous deletion CNV resulting in haploinsufficiency in human, restores brain development (Figure 5L-M) and suppresses the lethality of Ankle2A mutants (Figure 5N). Importantly, loss of one copy of ball (balle107) in Ankle2A mutants also restores the asymmetric protein localization of aPKC and Mira crescents in metaphase neuroblasts (Figure 5O-P). Hence, a partial reduction of Ball/VRK1 activity rescues Ankle2A mutants, providing strong evidence for a gene dosage sensitive locus. However, removing both copies of ball in wild type animals leads to pupal lethality (Cullen et al., 2005), causes severely reduced brain volumes in 3rd instar larvae (Herzig et al., 2014), and does not rescue Ankle2A animals, emphasizing that the gene dosage and balance of the protein levels is critical. Indeed, a severe loss of function allele, Ankle2CRIMIC, cannot be suppressed by reducing Ball/VRK1 activity (Figure 5N). In summary, these data demonstrate that both Ankle2 and Ball/VRK1 control the distribution of asymmetric determinants, and experimental evidence reveals an antagonistic relationship between both proteins.
The Ankle2-Ball pathway modulates aPKC and L(2)gl
Due to the similarities in defects observed with loss of Ankle 2 or aPKC, including mislocalization of Par6 and Mira (Kim et al., 2009), decreased cell divisions, and reduced neuroblast clone volume (Rolls et al., 2003), we hypothesized that the activity of aPKC, an important mediator of neuroblast asymmetric division (Figure 6A), might be affected. aPKC phosphorylation (Kim et al., 2009) or abundance could be modulated by Ankle2. We therefore assessed both total and phosphorylated aPKC levels in third instar larval brains using an antibody specific for human p-aPKC T410 (T422 in flies). This phosphorylation site is located in its activation loop and was shown to be important for its kinase activity (Kim et al., 2009). Phosphorylation of aPKC (T422) relative to total aPKC is decreased in Ankle2 mutants (Figure 6B) and is restored with either addition of Ankle2-GFPR or reduction of ball (Figure 6B), consistent with the data presented in Figure 5. However, overexpression of aPKC or constitutively active aPKC (aPKCΔN) (Betschinger et al., 2003) in Ankle2A mutants did not rescue brain size or viability (data not shown).
aPKC has been shown to physically interact with L(2)gl (Betschinger et al., 2003), a regulator of apico-basal polarity that inhibits the function of aPKC (Atwood and Prehoda, 2009; Wirtz-Peitz et al., 2008). aPKC and l(2)gl genetically interact as removal of one copy of aPKC suppresses l(2)gl loss of function phenotypes (Rolls et al., 2003), and aPKC has been shown to phosphorylate L(2)gl to control its plasma membrane or cortical release (Betschinger et al., 2003). When aPKC is active, L(2)gl is phosphorylated and released from the cortex; once released, it no longer binds to aPKC or inhibits its function. Because aPKC and L(2)gl interact, the Ankle2-Ball pathway may affect L(2)gl. We therefore assessed whether L(2)gl physically interacts with the Ankle2-Ball pathway using immunoprecipitation of a GFP-tagged L(2)gl from third instar larval brains and found that Ball indeed interacts with L(2)gl (Figure 6C).
The reduced aPKC activity that we observe may be associated with a gain of function of L(2)gl. Therefore, to determine whether removal of one copy of l(2)gl suppresses Ankle2 associated phenotypes, we introduced a temperature sensitive mutation of l(2)gl (l(2)glts3) into the Ankle2A mutant background and found that reducing l(2)gl in Ankle2A mutants at 22°C (Figure 6D-F) and 25°C (not shown) indeed partially restored brain size. Ankle2A is pupal lethal at 22°C, but when combined with a heterozygous l(2)gl mutant allele, some Ankle2A animals survive to adulthood. However, unlike the removal of one copy of ball, these animals die a few days after eclosion. In summary, Ankle2 and Ball interact with the apical-basal polarity regulators aPKC and L(2)gl (Figure 6A) and affect aPKC and L(2)gl activity by disturbing the asymmetric segregation of apical-basal polarity factors in neuroblasts.
Disease associated variants in VRK1 and its paralogs
Ten families have been described with biallelic variants in VRK1 that cause a spectrum of neurologic diseases including 6 individuals with microcephaly (Feng et al., 2018; Gonzaga-Jauregui et al., 2013; Najmabadi et al., 2011; Nguyen et al., 2015; Renbaum et al., 2009; Shaheen et al., 2018; Stoll et al., 2016) (Table S1; Figure S5). The family structures suggest either a sporadic or recessive neurological disease trait; historical consanguinity in 3/10 pedigrees implicate an autosomal recessive locus. Screening the BHCMG and BG databases identified two additional families with potentially biallelic variants in VRK1. (Table S2, Figure S5). These cases suggest that like ANKLE2 (Figure 1C and D), a heterogenous set of variant alleles in VRK1 are associated with neurologic disease and microcephaly.
It was previously shown that fly genes with more than one human homolog, especially those that are evolutionarily conserved, have an enriched association with OMIM disease phenotypes (Yamamoto et al., 2014). We searched the BHCMG database to establish if damaging variants in paralogs of VRK1 are associated with disease. Predicted deleterious, biallelic variants were found in two paralogs of VRK1: VRK2 is associated with very small eyes and VRK3 with severe microcephaly (Table S2, Figure S6).
NS4A targets the Ankle2 pathway
Drosophila has been developed as a model of viral infection (Harsh et al., 2018; Liu et al., 2018), and we recently showed that expression of the Zika protein NS4A results in reduced brain size in Drosophila. Strikingly, NS4A expression in Ankle2A/+ heterozygous animals leads to a more severe phenotype than NS4A expression in a wild type background, and these animals display brain phenotypes that mimic Ankle2CRIMIC null mutants (Shah et al., 2018). These data again suggest that levels of Ankle2 protein are critical, and hence, expression of NS4A in neuroblasts may cause microcephaly by affecting aPKC and Miranda localization. Indeed, expression of NS4A in neuroblasts (insc-GAL4>UAS-NS4A) affects the apical aPKC localization and leads to an expansion of the Mira domain at the basal membrane (Figure 7A-E). In the metaphase neuroblasts that express NS4A, we also note spindle orientation defects in some cells (Figure 7G-H), similar to Ankle2A animals shown in Figure 4. Hence NS4A targets the Ankle2 pathway; this is further strengthened with the observation that when NS4A is expressed in neuroblasts of ball heterozygous animals, aPKC and Mira crescents are restored to their wild type patterns (Figure 7D-F) and spindle orientation defects are rescued (compare Figure 7I with G and H). In summary, the Zika virus protein NS4A targets the Ankle2 pathway and affects asymmetric distribution of cell fate determinants, leading to defects in neuroblast division.
DISCUSSION
We investigated the biological basis for ANKLE2 associated microcephaly. We report six new patients with microcephaly that carry mutations in ANKLE2 and show that three variants identified in probands cause a loss of ANKLE2 function when tested in flies (Figure 1 and 2), providing compelling evidence that its loss causes reduced brain size in flies and severe microcephaly (Z-score < −2.5) in humans. Ankle2 is a dosage sensitive locus whose product is inhibited by the Zika virus protein NS4A. We show that Ankle2, like NS4A (Shah et al., 2018), is localized to the ER, and that it targets the nuclear envelope during mitosis. Loss of Ankle2 affects the nuclear envelope and ER distribution and results in a redistribution of Ball/VRK1, a kinase that is normally localized to the nucleus except when the nuclear envelope breaks down during mitosis (Figure 5). Loss of Ankle2 disrupts the localization of neuroblast apical-basal polarity determinants such as aPKC, Par 6, Baz, and Miranda, and aPKC phosphorylation is reduced by Ankle2 mutations. Importantly, loss of one copy of ball or l(2)gl suppresses the reduced brain volume associated with a partial loss of Ankle2, suggesting that much of the biological function of Ankle2 is modulated by aPKC and L(2)gl. Finally, the negative effect of NS4A on the activity of ANKLE2 can also be suppressed by removal of one copy of ball, suggesting the following pathway: NS4A ┤ ANKLE2 ┤ Ball/VRK1 → L(2)gl/LLGL1 ┤ aPKC. This represents an important pathway regulated by ANKLE2 that we show plays an important role in neuroblast stem cell divisions in flies and microcephaly phenotypes in humans.
Interestingly, the above pathway links environmental cues with several genetic causes of sporadic and AR microcephaly in human; moreover, it implicates this pathway in microcephaly accompanying congenital infection. As one example of the latter, the Zika virus has been shown to cross the infant Blood Brain Barrier (Mlakar et al., 2016) and has been identified in radial glial cells (Li et al., 2016), as well as intermediate progenitor 7 cells and neurons (Lin et al., 2017). We propose that NS4A affects the function of Ankle2 leading to the release of Ball/VRK1 from the nucleus. We speculate that this in turn affects the phosphorylation of aPKC and L(2)gl directly by masking phosphorylation sites or indirectly by promoting the activity of one or more phosphatases. Loss of VRK1 has been shown to cause microcephaly and some variant alleles are also associated with pontocerebellar hypoplasia (PCH) in humans (Gonzaga-Jauregui et al., 2013; Renbaum et al., 2009), consistent with the loss of ball in flies that causes a severe reduction in brain size (Yakulov et al., 2014). Note that ANKLE2, VRK1, LLGL1, and aPKC, as well as other components of the apical complex like PARD3 are all present in radial glial cells during cortical development (Ayoub et al., 2011). These data suggest that ANKLE2 and its partners such as LLGL1 and asymmetric determinants are important proteins during neural cell proliferation and that the proper levels and relative amounts of these proteins determine how many neurons will eventually be formed in vertebrates. Our data also indicate variant alleles at either ANKLE or VRK1 are responsible for some causes of embryonic lethality and severe congenital microcephaly.
LLGL1 has recently been shown to play an important role in radial glia in mice during neurogenesis, and its loss in clones increases the number of divisions (Beattie et al., 2017). In addition, aPKCζ/λ localizes at the apical membrane of proliferating neural stem cells in chicken embryos during division and has been shown to provide an instructive signal for apical assembly of adherens junctions (Ghosh et al., 2008). Mouse knockouts of aPKCλ (Soloff et al., 2004) and aPKCζ (Seidl et al., 2013) are embryonic lethal; however aPKCζ knockouts are viable (Leitges et al., 2001), perhaps suggesting redundant functions within the atypical PKC family. These proteins have not been linked to microcephaly in mice, but conditional removal of an apical complex protein Pals1 in cortical progenitors resulted in complete cortex loss (Kim et al., 2010). Finally, Numb is asymmetrically localized by the Par complex protein in Drosophila, segregated to the daughter cell during asymmetric cell division (Wirtz-Peitz et al., 2008), and essential for daughter cells to adopt distinct fates (Bhalerao et al., 2005). In mice, Numb localization is also asymmetric and null mutations exhibit embryonic lethality, neural tube closure defects, and premature neuron development (Zhong et al., 2000). These data indicate that asymmetric division may be important for vertebrate neuronal development, but microcephaly is not a phenotype that typically associates with loss of the mice homologues of asymmetric localized determinants studied Drosophila. However, the observations reported here indicate that the ANKLE2/PAR complex pathway is evolutionarily conserved from flies to human, although the precise mechanisms remain to be determined as different cells may use this pathway in different contexts (Suzuki and Ohno, 2006).
In order to determine whether predicted deleterious biallelic variants in PAR complex encoding genes or their paralogs associated with a neurologic disease trait, we searched the BHCMG database for mutations associated with neurological disease. We found homozygous predicted deleterious missense variants in PARD3B (c.1222G>A, p.G408S) in a patient that has microcephaly (Table S2, Figure S7) and compound heterozygous mutations in PARD3B (c.1654G>A;p.A552T) that are associated with other neurological defects (Table S2, Figure S7). The human orthologue of L(2)gl, LLGL1, is deleted in Smith-Magenis syndrome (SMS) (Smith et al., 1986) and 86-89% of the SMS patients have brachycephaly (Greenberg et al., 1996). These observations extend the mutational load beyond ANKLE2 and VRK1 and suggest an association between congenital disease and variants within the PAR complex (Table S2, Figure S7).
Aurora A (AurA) kinase has been shown to phosphorylate the Par complex (Wirtz-Peitz et al., 2008) as well as L(2)gl (Carvalho et al., 2015) and regulates cortical polarity and spindle orientation in neuroblasts (Lee et al., 2006). The aberrant localization of Ball/VRK1 in Ankle2 mutants may lead to gain of function phenotypes that are highly dosage sensitive, as they can be repressed by removing a single copy of Ball/VRK1 in Ankle2A. Mislocalized Ball/VRK1 may mask or interfere with the function of AurA in neuroblast asymmetric division as they share similar kinase substrate consensus sequences (Sanz-García et al., 2011; Ferrari et al., 2005). Future studies are needed to assess Ball/VRK1 redundancy or interference with AurA function.
Another possible evolutionarily parallel with implications in multicellular organismal development is the genetic interaction between the C. elegans homologue of VRK1 and an ANKLE2-like protein at the two cell stage (Asencio et al., 2012). Whereas VRK1 in both Drosophila and humans (Nichols and Traktman, 2004) is localized to the nucleus, except during mitosis when the nuclear envelope is broken down (Figure 5), the worm VRK1 protein is localized to the nuclear envelope. The worm ANKLE2-like protein, Lem-4L, also interacts with the phosphatase PP2A (Asencio et al., 2012), and Chabu and Doe (2009) noted that the fly PP2A regulates neuroblast asymmetric division by interacting with aPKC and excluding it from the basal cortex (Chabu and Doe, 2009). PP2A also antagonizes the phosphorylation of Baz by PAR-1 to control apical-basal polarity in dividing embryonic neuroblasts (Krahn et al., 2009) and regulates Baz localization in other cells such as neurons (Nam et al., 2007). This raises the possibility that the Ankle2 pathway also acts with PP2A in neuroblast asymmetric division.
Here, we identified a novel pathway that plays a significant role in neuroblast asymmetric division. By combining functional studies in Drosophila together with human subject data, we have linked several microcephaly-associated genes and congenital infection to a single genetic pathway. These studies allowed us to highlight conserved functions of the ANKLE2 pathway, and provide mechanistic insight on how a Zika infection might affect asymmetric division. This ANKLE2-VRK1 gene dosage sensitive pathway can be perturbed by genetic variants that disturb biological homeostasis resulting in neurological disease traits or by environmental insults such as Zika virus impinging on neurodevelopment. Hence, lessons learned from the study of rare diseases such as MCPH16/ANKLE2 can provide insights into more common disease and potential gene by environmental interactions.
Author Contributions
N.L. and H.J.B. conceived the project and designed experiments, and wrote and revised the manuscript with J.R.L. H.C. performed in vivo Ankle2 immunoprecipitations and assessed phosphorylation/total protein levels. A.J. assisted with brain volume measurements. A.J., M.W. and J.R.L performed primary fibroblast experiments, and human mutation studies. H.A., B.B.G., T.T., S.I., B.T., G.M.M., G.H.M., A.X.J., and R.D.C. ascertained clinical and molecular data of children with variants. B.T., B.A., P.S., and N.K. assisted with ZIKV experiments. N.L. performed all other experiments.
Methods
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hugo J. Bellen (hbellen{at}bcm.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Drosophila melanogaster
The following fly lines were used: FRT19a (Yamamoto et al., 2014), Ankle2A (Yamamoto et al., 2014), Ankle2CRIMIC (this study), Ankle2IGFP(this study), Ankle2-GFPR (this study), P{UASt-hANKLE2}VK37 (Yamamoto et al., 2014), P{UASt-hANKLE2 p.L537V}VK37 (this study), P{UASt-hANKLE2 p.Q782*}VK37 (this study), P{UASt-hANKLE2 p.A109P}VK37 (this study), P{UASt-hANKLE2 p.G201W}VK37 (this study), P{20XUAS-tdTomato-Sec61beta}attP2 (Summerville et al., 2016), balle107(Cullen et al., 2005), ball2 (Herzig et al., 2014), l(2)glMI07575-GFSTF.0 (Nagarkar-Jaiswal et al., 2015a), l(2)glts3cn1sp13 (Manfruelli et al., 1996), P{UASt-NS4Aug} (Shah et al., 2018), P{UASt-CD8-GFP} (Lee and Luo, 2001), Actin-GAL4 (P{Act5C-GAL4}17bFO1) (Ito et al., 1997), inscuteable-GAL4 (P{w[+mW.hs]=GawB}insc[Mz1407]) (Luo et al., 1994), daughterless-GAL4 (P{w[+mW.hs]=GAL4-da.G32}UH1) (Wodarz et al., 1995), ball-GFP (fTRG-823) (Sarov et al., 2016), wor-mira-cherry P{w[+mC]=wor.GAL4.A}2,P{w[+mC]=UAS-mira.cherry}2/CyO) (Cabernard and Doe, 2009), P{His2Av[T:Avic\GFP-S65T]}62A (Clarkson and Saint, 1999), P{w[+mC]=UAS-aurA.Exel}2, M{ UAS-aPKC.ORF.3xHA}ZH-86Fb (Bischof et al., 2013), P{w[+mC]=UAS-aPKC.DeltaN}3 (Drier et al., 2002). All flies were maintained at 22°C and grown on standard cornmeal and molasses medium in plastic vials. Crosses were performed at temperature indicated (18°C, 22°C, 25°C, or 29°C). Hemizygous males were analyzed as Ankle2 mutants (which is on the X chromosome) and females were used for Ankle2 heterozygous studies. All other studies contained males and females. Brain volume measurements were conducted in late 3rd instar larvae (gut clearance, extruding spiracles). Act-GAL4 was use to ubiquitously express ZIKV NS4A and Sec61β, da-GAL4 was used to express human ANKLE2 constructs and aPKC, insc-GAL4 was used to express NS4A in neuroblasts.
Human studies
All study subjects enrolled into the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) provided informed consent for exome sequencing and study participation under the Baylor College of Medicine Institutional Review Board-approved protocol H-29697. BAB701 provided informed consent for molecular and genomic analysis under the Baylor College of Medicine Institutional Review Board-approved protocol H-9170. All study subjects enrolled through Baylor Genetics Laboratory (BGL) were analyzed on a retrospective basis, and only de-identified information is provided under the Baylor College of Medicine Institutional Board approved protocol H-41191. Patients were ascertained from the 7148 sequenced individuals in BHCMG or the ∼12500 sequenced individuals in the BGL by searching for biallelic variants with CADD scores >15 in conjunction with phenotypes of interest. Six male and two female patients were ascertained from the BHCMG database. The age of the patient is known for 3 individuals (2y, 7y, 32y). Two female and three male patients were ascertained from the BGL database. Ages at referral were 2 months, 7y, 12y, 20y, and 41y.
Human primary cultures
Fibroblasts were cultured in flasks containing Gibco DMEM (1x) with 4.5g/L D-Glucose, L-Glutamine, 25mM HEPES, HyClone FBS (10%), and Gibco Anti-Anti (1%) at 37 degrees Celsius with 5% CO2. Cultures of p.L573V/+ and p.L573V/p.Q782* were male; p.V229G/p.V229G was female.
METHOD DETAILS
Generation of Ankle2 mutations and constructs
To generate Ankle2CRIMIC by CRISPR-Cas9, two guide RNAs targeting Ankle2, 5’-ATAAAGTATTTTCTTAACGGTGG-3’ and 5’-TAATAATTTTAAATTCTCATTGG-3’ with PAM sites underlined, were cloned into pCDF3 (Port et al., 2014). Regions of homology targeting the 4th coding intron were cloned into PM14 (Lee et al., 2018) using 5’-ccatagctatggGCAATTCCTCAATGTCGAATTTACTGCTCA-3’ and 5’-ttatgcatATTTTCTTAACGGTGGGAAATTATAC-3’ to amplify the left arm for BstXI/NsiI cloning and 5’-tagcatgcATACTTTATTATTGCATTTGTTATAAGTATGAGA-3’ and 5’-tactcgagGCAAAGTTCCAGACCGTTTCTGATTTATC-3’ to amplify the right arm for conventional cloning with SphI/XhoI. This donor construct and two guide RNA constructs were injected into, w;attP40(y+){nos-Cas9(v+)}/CyO (Kondo and Ueda, 2013) embryos, and positive expression of 3XP3-GFP was used to isolate animals with targeted events. PCR and genomic sequencing of surrounding regions validated the Ankle2CRIMIC allele. AnkleIGFP was generated by RMCE by injecting a plasmid expressing integrase with pBS-KS-attB1-2-PT-SA-SD-EGFP-FlAsH-StrepII-TEV-3xFlag (Nagarkar-Jaiswal et al., 2015b) into Ankle2CRIMIC animals. Animals with 3XP3-GFP loss were screened using PCR for targeted cassette exchange. Regions flanking the targeted event were sequenced to verify the allele. The Ankle2-GFPR was created using recombineering (Venken et al., 2008) of BAC CH321-85N12 (Venken et al., 2006). A GFP donor construct was generated by amplifying the GFP coding region and a selection cassette from plasmid PL-452 C-EGFP with primers containing 50bp homology with the C-terminal end of Ankle2 (5’-GGGATCAACGGTCCTATAACGAGGGGGACACGCCGCTGGGCAATCGGAACGCAGCCCAATTCCGATCATATTC-3’ and 5’-CATCAATCAGTCGCTGTTTCTGTTTCTGTTTCCGGGCCGATT CCGTTTCATTACTTGTACAGCTCGTCCATG-3’. Regions matching PL-452 C-EGFP are underlined. CH321-85N12 was transformed into DY380 cells using electroporation. Stable colonies were grown overnight at 30°C, induced for recombination functions at 42°C for 15 min, and transformed using electroporation (1.8kV, 200Ohm, 25μFD) with the amplified donor construct. Colonies were selected for both the BAC (chloramphenicol) and insertion of GFP (kanamycin). Resulting colonies were verified using PCR, restriction enzyme digestion, and sequencing. The GFP tag selection cassette was removed using Cre mediated excision by transforming the Ankle2-GFP BAC into induced EL350 cells. Properly excised events were verified by PCR, absence of growth on kanamycin selection plates, and sequencing.
Generation of human ANKLE2 expression constructs
NEB Q5 Site-directed mutagenesis was performed on P{UASt-hANKLE2}. Each plasmid was sequence verified and injected into VK37 flies with a plasmid expressing integrase for site-specific integration.
Brain immunostaining
Late 3rd instar (based on gut clearance and extruding spiracles) larval brains were dissected in PBS and fixed with 4% PFA/PBS/0.3%Triton for 20 minutes. For immunostaining, brains were blocked in PBS/0.3%Triton/1%BSA/5% normal goat serum and incubated in primary antibody in PBS/0.3%Triton/1%BSA overnight. Primary antibodies include rat anti-Deadpan (Abcam Cat# ab195172, 1:250 or 1:500), mouse anti-Prospero MR1A (Developmental Studies Hybridoma Bank, 1:1000), rat anti-Miranda (1:500, Abcam Cat# ab197788), rabbit anti-aPKC (1:1000, PKCz (C-20) Santa Cruz, discontinued), rabbit anti-GFP (1:1000, Invitrogen Cat# A11122), mouse anti-Calnexin 99a (Developmental Studies Hybridoma Bank, 1:100), mouse anti-Lamin Dm0 ADL67.10 (Developmental Studies Hybridoma Bank, 1:250), guinea pig anti-Bazooka (1:1000) (Siller et al., 2006), rat anti-Par6 (1:50) (Rolls et al., 2003), rabbit anti-phospho-Histone H3 (1:1000, Millipore Cat# 06-570), rabbit anti-Ball (1:1000) (Yakulov et al., 2014), rabbit anti-VRK1 (1:1000, Abcam Cat# ab151706), rabbit anti-CNN (1:1000) (Lucas and Raff, 2007), and mouse anti-Strep (Qiagen Cat# 34850, 1:500) with goat or donkey secondary antibodies from Jackson ImmunoResearch used 1:500. Brains were mounted with double sided tape spacers and imaged using a Leica Sp8 with 2 µm or 3 µm sections through the entire brain lobe.
Live imaging
3rd instar larvae were dissected in sterile PBS supplemented with 1% FBS and 0.5mM ascorbic acid, fine dissected on an inverted Sarstedt lumox dish 50 in a petroleum jelly well. Samples were imaged on a Leica Sp8 with optimized settings for high quality images without bleaching or a Zeiss 880 with Airy scan (wild type Ankle2-GFP and Sec61β colocalizaiton).
Protein immunoprecipitation, mass spectrometry, and western analysis
3rd instar larvae or dissected larval brains from l(2)glMI07575-GFSTF.0 animals were dissociated in 0.1% CHAPS buffer supplemented with protease and phosphatase inhibitors for at least 30 min on ice, centrifuged for 10 min at 4°C, and supernatant was used for immunoprecipitation or western analysis. For immunopreciptation, 25ul of Allele Biotechnology GFP nanoantibody agarose (nAb, Cat# ABP-NAB-GFPA100) was equilibrated and incubated with lysate 2hrs - overnight at 4°C with rotation. Agarose was spun down for 1 min at 1000 × g at 4°C, supernatant was removed, and pellet was washed 3X (1X binding buffer (10mM Tris-HCl pH 7.5,150mM NaCl), 2X wash buffer (10mM Tris-HCl pH7.5, 500mM NaCl)). Remaining agarose pellet was submitted to MD Anderson Proteomics and Metabolomics Facility core for mass spectrometry target identification, or eluted for western analysis in loading buffer. For western analysis, larval brains were dissected and dissociated as stated above, and were lysed in 0.1% CHAPS buffer [[50mM Nacl, 200mM HEPES, 1mM EDTA and protease inhibitor cocktail (Roche)] Loading input was adjusted for brain size and protein concentration. Primary antibodies include rabbit anti-GFP (1:2500, Invitrogen Cat# A11122), rabbit anti-Ball (1:1000) (Herzig et al., 2014), rat anti-L(2)gl (Peng et al., 2000), rabbit anti-aPKC c-20 (1:1000, Santa Cruz, discontinued), rabbit anti-aPKC phosphoT410 (1:1000, Santa Cruz, discontinued), and mouse anti-Actin-c4 (1:5000, Millipore Cat# MAB1501). Secondary antibodies include Rockland DyLight 600 and 800 (1:1000), BioRad Star Bright Blue 700 (1:1000) and Jackson ImmunoResearch HRP conjugated (1:5000). Blots were imaged on a Bio-Rad ChemiDocMP.
Human cell immunohistochemistry
Cells were detached using Trypsin-EDTA 0.05% and plated onto 18mm glass cover slips in 6 well plates. Cells were cultured for an additional 3 days under the same conditions before fixing and staining. Cells were rinsed with PBS followed by fixing in 4% paraformaldehyde in PBS. Cells were rinsed and washed 3x in PBST, washed 2x in PBST + 1% BSA (PBSTB), and then blocked in PBSTB + 5% normal goat serum. They were then incubated in PBSTB and primary antibody overnight at 4 degrees Celsius. Cells were then washed 3x in PBSTB, incubated in anti-rabbit Cy5 secondary antibody (1:500) for 2 hours, and washed 3x in PBST. The cells were given a final wash in PBST with DAPI (1:1000) for 30m before mounting using SlowFade glow on glass slides and sealing with nail polish.
Exome and Sanger Sequencing
Exome sequencing was performed under the Baylor Hopkins Center for Mendelian Genomics (BHCMG) research initiative as previously described (Lupski et al., 2013). Exome capture was performed with Nimblegen reagents and a custom capture reagent, VCRome2.1. Raw data was processed using the Mercury pipeline, available on DNANexus (http://blog.dnanexus.com/2013-10-22-run-mercury-variant-calling-pipeline/) (Reid et al., 2014) and the ATLAS2 method was used for variant calling followed by an in-house Cassandra annotation pipeline based on Annotation of Genetic Variants (ANNOVAR). The LLGL1 variant was orthogonally validated and segregated with disease by dideoxy Sanger sequencing of PCR amplicons (Sanger et al., 1977).
QUANTIFICATION AND STATISTICAL ANALYSIS
Brain volume
Brains from third instar larvae were stained and mounted with tape spacers and imaged using a Leica Sp8 with 2 µm or 3 µm sections through the entire brain lobe. Resulting stacks were analyzed using the Surfaces function in Imaris (Bitplane) to quantify brain lobe volume as total microns cubed. One lobe from each brain was imaged and a total of 5-10 brains were analyzed per genotype or condition. Brain lobe volumes are displayed as box plots with hinges representing the 25th to 75th percentiles, a line represents the median, and whiskers represent min to max. Statistical significance was determined using one-way ANOVA with multiple comparisons post-test calculated using GraphPad Prism. Brain volumes in Figure 1 are normalized to wild type (FRT19a). Average volume from wild type is set to 100%, and each mutant or condition is normalized as percentage of wild type volume. Brain volumes from Figure 4-6 are displayed as total brain volume (μm 3).
Asymmetric phenotypes
3rd instar larvae were immunostained for pH3, Baz, Par6, aPKC, or Mira as described above. Metaphase neuroblasts (pH3 positive, chromosomes aligned at the metaphase plate) were imaged on a Leica Sp8 (63X). Only metaphase neuroblasts in the correct plane for imaging were analyzed. Mild disruption refers to weak or incomplete crescent localization, and strong disruption indicates no crescent localization. To quantify spindle orientation, CNN was used to mark the plane of division, and aPKC, and Mira were used to establish cortex polarity. Only metaphase neuroblasts in the correct plane for imaging were analyzed. The angle between spindle orientation and cortex polarity was measured using the angle function of ImageJ. Phenotypes are portrayed as percentage of total counted metaphase neuroblasts. For all samples n>20 but <60.
VRK1 intensity
Human fibroblasts were stained as described above, imaged on a Zeiss 710 as Z-stacks with equivalent laser power and confocal settings in the same imaging session. Resulting images were analyzed in Imaris (Bitplane) using the Surfaces function to mark nuclear volume. Total intensity sum of the VRK1 channel within the nucleus and nuclear volume were recorded. VRK1 intensity is displayed as intensity sum normalized to volume. One-way ANOVA with multi-comparisons post-test from GraphPad Prism was used to assess significance. Each dot represents one nucleus. Three fields from each cell line were assessed.
Supplemental Tables
Table S1: Summary of ANKLE2 and VRK1 published cases. Related to Figure 1 and 5.
Published patient variants in ANKLE2 and VRK1 with associated phenotypes.
Table S2: Summary of ANKLE2, VRK1, PAR complex, and LLGL1 variants in BGL and BHCMG databases. Related to Figure 1, 5, and 6. Unpublished human variants in the ANKLE2 pathway with analysis of variants. Zyg: Zygosity; vR/tR: Variant Reads to Total Reads Ratio; phyloP: evolutionary conservation score; ARIC: frequency in Atherosclerosis Risk in Communities database; CADD: Combined Annotation Dependent Depletion Score; gnomAD: frequency of heterozygotes and homozygotes in the publicly available gnomAD database; AOH block: Absence of Heterozygosity regions in which homozygous variants were found.
Supplemental Movies
Movie S1: Ankle2-GFPR localizes to the nuclear envelope during division. Related to Figure 3. Time lapse images of live 3rd instar larval brains from Ankle2-GFPR animals showing subcellular localization of Ankle2 protein (green). Large cells are neuroblasts. Ankle2 is cytoplasmic during interphase but is recruited to the nuclear envelope during cell division.
Movie S2: Ankle2-GFPR localizes to the nuclear envelope during division. Related to Figure 3. Time lapse images of live 3rd instar larval brains from Ankle2-GFPR animals showing subcellular localization of Ankle2 protein (green). Large cells are neuroblasts. Ankle2 is cytoplasmic during interphase but is recruited to the nuclear envelope during cell division.
Movie S3: Miranda localizes to the basal membrane in wild type dividing neuroblasts. Related to Figure 4. Time lapse imaging of live 3rd instar larval brains from wild type animals expressing Miranda-GFP (green) and Histone-RFP (DNA, white). Large cells are neuroblasts. Note the localization of Miranda at the basal membrane during mitosis.
Movie S4: Ankle2A mutant neuroblasts fail to segregate DNA. Related to Figure 4. Time lapse imaging of live 3rd instar larval brains from Ankle2A animals expressing Miranda-GFP (green) and Histone-RFP (DNA, white). Large cells are neuroblasts. Note reduced localization of Miranda at the basal membrane and DNA segregation failures during mitosis.
Movie S5: Ankle2A mutant neuroblasts fail to undergo cytokinesis. Related to Figure 4. Time lapse imaging of live 3rd instar larval brains from Ankle2A animals expressing Miranda-GFP (green). Large cells are neuroblasts. Note reduced localization of Miranda at the basal membrane and cytokinesis failure during mitosis.
Movie S6: Ankle2A mutant neuroblasts have defective Miranda localization. Related to Figure 4. Time lapse imaging of live 3rd instar larval brains from Ankle2A animals expressing Miranda-GFP (green) and Histone-RFP (DNA, white). Large cells are neuroblasts. Note reduced localization of Miranda at the basal membrane.
Movie S7: Ballchen localizes to the nucleus during interphase and redistributes throughout the entire cell during mitosis. Related to Figure 5. Time lapse imaging of live 3rd instar larval brains from wild type animals expressing Ballchen-GFP (green). Large cells are neuroblasts. Note the nuclear localization of Ball during interphase, redistribution during mitosis, and recruitment back to the nucleus upon completion of mitosis.
Acknowledgements
We would like to thank many members of the Bellen lab for suggestions, and Karen Schulze for critical reading. We thank Chris Doe, Jurgen Knoblich, Jim Skeath, Kenneth Prehoda, Alf Herzig, Hiroyuki Ohkura, and the Bloomington Drosophila Stock Center for providing stocks and reagents, and the IDDRC Microscopy Core (NIH/NICHD U54 HD083092) for valuable input. This work was supported by NIH/NINDS F32NS092270 to N.L., Howard Hughes Medical Institute (HHMI) Medical Research Fellowship to A.J., the NIH/NINDS K08NS092898 and Jordan’s Guardian Angels to G.M.M., a jointly funded NHGRI and NHLBI grant to the Baylor-Hopkins Center for Mendelian Genomics (UM1 HG006542) and NIH/NINDS R35NS105078 to J.R.L, NIH U54NS093793, NIH R24OD022005, and the Huffington Foundation to H.J.B.. N.L. and H.C. are supported by HHMI, and H.J.B. is an Investigator of the Howard Hughes Medical Institute.