Abstract PPFIA3 encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin- α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.
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.
Abstract Notch pathway is an evolutionarily conserved signaling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. To identify novel effectors of Notch signaling, we analyzed the whole transcriptome of Drosophila wing and eye imaginal discs in which an activated form of Notch was overexpressed. A LIM homeodomain protein Arrowhead (Awh) was identified as a novel candidate which plays a crucial role in Notch mediated developmental events. Awh alleles show strong genetic interaction with Notch pathway components. Awh loss-of-function upregulates Notch targets Cut and Wingless. Awh gain-of-function downregulates Notch targets by reducing the expression of ligand, Delta. Consequently, the expression of Wingless effector molecule Armadillo and its downstream targets, Senseless and Vestigial, also gets downregulated. Awh overexpression leads to ectopicexpression of engrailed , a segment polarity gene in the anterior region of wing disc, leading to patterning defects. Additionally, Notch gain-of-function mediated neuronal defects get significantly rescued with Awh overexpression. Activated Notch inhibits Awh activity, suggesting a regulatory loop between Awh and Notch. Additionally, the defects caused by Awh gain-of-function were remarkably rescued by Chip, a LIM interaction domain containing transcriptional co-factor. The present study highlights the novel feedback regulation between Awh and Notch.
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. 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.
Notch signaling is an evolutionarily conserved pathway that is found to be involved in a number of cellular events throughout development. The deployment of the Notch signaling pathway in numerous cellular contexts is possible due to its regulation at multiple levels. In an effort to identify the novel components integrated into the molecular circuitry affecting Notch signaling, we carried out a protein-protein interaction screen based on the identification of cellular protein complexes using co-immunoprecipitation followed by mass-spectrometry. We identified Hrp48, a heterogeneous nuclear ribonucleoprotein in Drosophila, as a novel interacting partner of Deltex (Dx), a cytoplasmic modulator of Notch signaling. Immunocytochemical analysis revealed that Dx and Hrp48 colocalize in cytoplasmic vesicles. The dx mutant also showed strong genetic interactions with hrp48 mutant alleles. The coexpression of Dx and Hrp48 resulted in the depletion of cytoplasmic Notch in larval wing imaginal discs and downregulation of Notch targets cut and wingless Previously, it has been shown that Sex-lethal (Sxl), on binding with Notch mRNA, negatively regulates Notch signaling. The overexpression of Hrp48 was found to inhibit Sxl expression and consequently rescued Notch signaling activity. In the present study, we observed that Dx together with Hrp48 can regulate Notch signaling in an Sxl-independent manner. In addition, Dx and Hrp48 displayed a synergistic effect on caspase-mediated cell death. Our results suggest that Dx and Hrp48 together negatively regulate Notch signaling in Drosophila melanogaster.
Summary Owing to a multitude of functions, there is barely a tissue or a cellular process that is not being regulated by Notch signaling. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. To identify novel effectors of Notch signaling in Drosophila melanogaster , we analyzed the whole transcriptome of the wing and eye imaginal discs in which an activated form of Notch was overexpressed. Selected candidate genes from the transcriptome analysis were subjected to genetic interaction experiments with Notch pathway components. Among the candidate genes, T‐box encoding gene, Dorsocross ( Doc ) showed strong genetic interaction with Notch ligand, Delta . Genetic interaction between them resulted in reduction of eye size, loss of cone cells, and cell death, which represent prominent Notch loss of function phenotypes. Immunocytochemical analysis in Df(3L)DocA/Dl 5f trans‐heterozygous eye discs showed accumulation of Notch at the membrane. This accumulation led to decreased Notch signaling activity as we found downregulation of Atonal, a Notch target and reduction in the rate of Notch‐mediated cell proliferation. Doc mutant clones generated by FLP‐FRT system showed depletion in the expression of Delta and subsequent reduction in the Notch signaling activity. Similarly, Doc overexpression in the eye discs led to modification of Delta expression, loss of Atonal expression and absence of eye structure in pharate adults. Taken together, our results suggest that Doc regulates the expression of Delta and influence the outcome of Notch signaling in the eye discs.
