TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities

Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling

Abstract Objectives Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble (scrib), lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. Our study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context. Results In the present study, we use Drosophila as an in vivo model to show the synergy between activated Notch (N act ) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of N act and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. We further show activation of JNK signaling and upregulation of its receptor, Grindelwald in N act /lgl-IR tumor. In contrast to previously described Notch act /scrib −/− tumor, our experiments in N act /lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth

Pleiotropic Functions of the Chromodomain-Containing Protein Hat-trick During Oogenesis in Drosophila melanogaster

Chromatin-remodeling proteins have a profound role in the transcriptional regulation of gene expression during development. Here, we have shown that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome, and that a mild expression is observed in follicle cells. Colocalization of Hat-trick with Heterochromatin Protein 1 and synaptonemal complex component C(3)G along with the diffused karyosome after hat-trick downregulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. Germline mosaic analysis reveals that hat-trick is required for maintaining the dorso-ventral patterning of eggs by regulating the expression of Gurken. The increased incidence of double-strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility, and, consequently, misexpression and altered localization of Gurken in hat-trick mutant egg chambers clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, we speculate a substantial role of hat-trick in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. Our results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster

MOESM1 of Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling

Additional file 1. Materials and methods

MOESM3 of Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling

Additional file 3: Figure S2. N act /lgl-IR tumor leads to distorted actin cytoskeleton. Coexpression of N act and lgl-IR causes distorted actin cytoskeleton organization (d) compared to that of wild-type (a), only N act overexpressed (b) and only lgl-IR overexpressed condition (c). F-actin was marked using phalloidin. Scale bars: 10 ¾m (a-d)

MOESM5 of Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling

Additional file 5: Figure S4. Lowering the dose of Notch partially rescues lgl-IR-induced MMP1 expression and restores the adult wing. a MMP1 expression in wild-type wing disc is shown. b Overexpression of only Notch-DN did not induce expression of MMP1. c Overexpression of lgl-IR induces MMP1 expression in the wing disc. d Coexpression of Notch-DN in lgl-IR background partially rescues the expression of MMP1 caused by lgl-IR overexpression. a′, b′, c′ and d′ are merges of DAPI along with a, b, c and d, respectively. Moreover, Coexpression of Notch-DN with lgl-IR resulted in reduced wing disc size as compared to that of only overexpression of lgl-IR (i). e GFP marked vestigial domain in wing disc is shown. e′ is the merge image of DAPI along with (e). f Overexpression of Notch-DN resulted in held out wings with wing nicking phenotype. g Overexpression of lgl-IR using vg-GAL4 led to necrotic lesions followed by deformation of adult wings. h Coexpression of Notch-DN with lgl-IR partially restored deformed adult wings. j Phenotype penetrance in adult flies is shown for each genotype; the phenotype observed in Notch-DN show 100% penetrance and around 70% lgl-IR flies showed deformed wings. In case of Notch-DN; lgl-IR flies, around 60% flies showed the depicted phenotype and, the rest of the flies showed less developed wings but they were not of the lgl-IR category. Analysis of data was done using One-way ANOVA with Tukey’s multiple comparison test; data represents mean ± SEM (***p < 0.001 and ns p > 0.05). All wing discs are oriented with dorsal to the top and posterior to the right. Scale bar: 50 µm (a–d, a′–d′)

MOESM2 of Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling

Additional file 2: Figure S1. Quantification of GFP and MMP1 in the VNC of N act /lgl-IR tumor (a) GFP quantification in VNC shows a four-fold increment in the amount of GFP positive cells in N act /lgl-IR as compared to that of the wild-type, only N act and lgl-IR overexpressed tissues. b MMP1 quantification in VNC shows around four-fold increase in N act /lgl-IR, whereas only N act and lgl-IR overexpressed tissues show almost same level of MMP1 in VNC as of wild-type. c Real-Time PCR analysis shows significant increase in mmp1 transcripts in the cephalic complex of N act /lgl-IR as compared to that of wild-type, only N act or only lgl-IR tissues. Data was normalized to rps17. Analysis of data was done using One-way ANOVA with Tukey’s multiple comparison test; data represents mean ± SEM (***p < 0.001 and ns p > 0.05)

MOESM6 of Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling

Additional file 6: Figure S5. Inhibition of JNK pathway suppresses the N act /lgl-IR tumor growth and MMP1 expression. Fluorescent micrographs of wing imaginal discs are shown. a Overexpression of both N act and lgl-IR in wing imaginal disc using vg-GAL4 resulted in massive upregulation of MMP1. b Coexpression of bsk DN in the background of N act and lgl-IR resulted in the suppression of MMP1 expression. a″–b″ is the merge images of a–a′ and b–b″. c The N act /lgl-IR wing disc size was significantly reduced, when bskDN was expressed in the background. Analysis of data was done using Unpaired t test with Welch’s correction; data represents mean ± SEM **p < 0.01). All wing discs are oriented with dorsal to the top and posterior to the left. Scale bar: 50 µm (a–a″, b–b″)

De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment

The Translocase of Outer Mitochondrial Membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyperreflexia, ataxia, dystonia, and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1, was found to have a p.Thr607Ile variant while individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4 driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knock-down of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals