The Wnt signaling network has critical roles in embryonic development and is implicated in human disease. One of the outputs of the Wnt network, called the planar cell polarity (PCP) pathway, regulates tissue polarity and directs cell migration. Core PCP components (Frizzled, Dishevelled, Prickle, Vangl, Celsr) localize asymmetrically in polarized cells and establish polarity across the tissue through protein interactions between adjacent cells. The core PCP component activate tissue-specific "effectors" which translate the signal into morphological changes. PCP is related to several disease conditions, including neural tube defects, cystic kidney disease, and cance metastasis. However, mechanisms of the PCP underlying physiological and disease-related conditions are not well understood. Here, I explore functions of the core PCP component Pk, and its relationship to disease, in the zebrafish model system. Mutations in Pk1 and Pk2 have been identified in human progressive myoclonic epilepsy patients. Pk coodinate cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet, how dysfunctions of pk relates to epilepsy is unknown. Here, I show that knockdown of pk1a sensitizes the zebrafish larva to convulsant drug. To model the defects in central nervous system, I examine neurogenesis in the retina and find that both pk1a and pk2 are required for proper dendritic outgrowth in the retinal inner plexiform layer. Furthermore, I characterize the epilepsy-related mutant forms of Pk1a and Pk2. The mutant Pk1a forms show reduced ability to suppress the retinal neurogenesis defects compared to the wild-type, as well as differential ubiquitination levels. Pk2 mutant forms also show differential activities in overexpression assays and seemingly more stable proteins relative to the wild-type. Taken together, pk1a and pk2 may contribute to epilepsy by affecting neuronal patterning and thus signal processing. Another aspect of PCP function has been implicated in cilia and cilia-related disorders, also called ciliopathy. PCP effectors have been shown to modulate ciliogenesis and core PCP proteins (Vang and Dvl) regulate cilia orientation. On the other hand, cilia are not required for PCP signaling, especially asymmetric core PCP protein localization. These findings leave open the question what is the precise relationship between PCP and cilia. The Bardet Biedl Syndrome (BBS) is a type of ciliopathy that leads to obesity, retinitis pigmentosa, polydactyly, mental retardation and other symptons. A subset of BBS genes share similar knockdown phenotype in cell migration as seen in PCP knockdown embryos. Shared pehnotypes have led some to proposethat PCP and bbs genes may interact. Yet a direct relationship has yet to be established. I examine the interaction between pk2 and a central Bbs gene, bbs7. By analyzing shared phenotypes in double knockdown embryos, I find no synergistic interaction between the two, suggesting they act in distinct pathways. Bbs regulate ciliary trafficking and in zebrafish, knockdown of bbs genes leads to delayed retrograde melanosome transport. Interestingly, I find knockdown of pk2 suppresses this retrograde transport delay. Additionally, pk2 knockdown embryos show a delay in anterograde melanosome transport. These findings highlight a new role for pk2 in intracellular transport and clarifies the relationship between PCP and BBS. In summary, my work here strengthens the link between pk mutations and human epilepsy and identifies functions of pk in retinal neurogenesis and in intracellular transport. To what extent the role of neurogenesis and intracellular transport are related is worth future study. Yet, this new information provides insights into potential mechanisms of epilepsy and the relationship between PCP and BBS.

About 5% to 10% of epileptic patients suffer from Juvenile Myoclonic Epilepsy (JME), which is characterized by spasms of the arms, ataxia (uncoordinated movements), and general tonic-clonic seizures. In a recent study, a group of patients with myoclonic epilepsy was found to harbor mutations in the PRICKLE1 and PRICKLE2 genes. This suggested that PRICKLE genes might be linked to epilepsy, and given that PRICKLE is highly evolutionarily conserved (including in fruit flies), we decided to use Drosophila in order to determine, first, whether flies with prickle mutations were seizure-prone, and if so, to then use the powerful genetic tools of Drosophila to elucidate the underlying mechanism of the prickle-associated epilepsy. In this work, we show that mutation of the pksple isoform (one of the two adult prickle isoforms in flies) lowers the seizure threshold in the mutant flies (resulting in seizure activity), while mutation of the other adult isoform, pkpk, had no effect. This was demonstrated through both behavioral assays (where the pksple mutant flies showed a reduction in recovery of climbing behavior after being subjected to mechanical stimulation while the pkpk mutant flies did not) as well as electrophysiological analysis (where pksple mutants were shown to be hyperexcitable after electrical stimulation, while the pkpk allele showed no change in spiking activity). We demonstrated that the underlying mechanism of the hyperexcitability seen in the pksple flies was due to enhanced anterograde transport on microtubule (MT) tracks in neurons, the main route for transport in neurons, which could be suppressed by reducing the dose of either of two Kinesin motor proteins, the motors involved in anterograde transport in neurons. On the other hand, the pkpk mutants showed the reverse effect, exhibiting a significant reduction in vesicle transport dynamics. We showed that microtubule polarity could be partially reversed by tipping the balance of the pk isoforms similar to what is seen in the pkpk mutants (such that a large percentage of MTs now had their plus ends oriented towards the cell body, which is extremely rare in axons), suggesting that the vesicle transport defects seen in the pkpk mutants might be due to mixed polarity of MTs. Next, we showed that the seizure-prone pksple mutants, but not the pkpk mutants, exhibited a myoclonic form of epilepsy, as well as abnormal walking patterns and uncoordinated movements, paralleling the ataxia phenotype seen in the epileptic patients with PRICKLE mutations. These data suggest that the primary aspects of the epilepsy-ataxia syndrome seen in patients with PRICKLE mutations are recapitulated in flies, which underscores the utility of using the fruit fly genetic system to model this disorder. Finally, our preliminary results suggest that the pk alleles have different effects on neuronal morphology due to changes in sizes of terminal boutons at the neuromuscular junction (NMJ) in larvae. These data suggest that pk is having a direct effect on synaptic formation and likely function. In conclusion, by using our Drosophila model system, we were able to link prickle mutations to epilepsy as well as identify the cellular mechanism of the prickle-associated epilepsy, a novel epilepsy mechanism previously associated with neurodegeneration. To our knowledge, this is the first example of a gene that, when mutated, will cause seizures in flies, zebrafish, mice, and humans, indicating that the role of prickle in controlling seizure activity is remarkably conserved in animals. Significantly, since about one third of patients with epilepsy do not respond to current AEDs, our fly model and the techniques we have developed will enable us to conduct drug screens for testing potential chemical compounds as new AEDs.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

We sought to investigate the role of the Planar Cell Polarity (PCP) pathway in neuronal positioning along the Anterior-Posterior (AP) axis of C. elegans, and chose the worm’s DD-type motor neurons as a model. The six DD neurons (DD1-DD6) are evenly spaced in the ventral nerve cord of wild type animals. Here we showed that mutations in core PCP genes caused DD neuron spacing and positioning defects. prkl-1 double mutant combinations with vang-1 and fmi-1 showed a suppression of the more severe prkl-1 single mutant defects, which was evidence of genetic interactions between these PCP components. We also conducted a candidate screen of Frizzled, Dishevelled, Wnt, and ROCK genes, and found that dsh-1/Dishevelled, mom-2/Wnt and let-502/ROCK also played roles in DD neuronal positioning. Both vang-1 and prkl-1 were found to function within the nervous system to guide DD neuronal positioning, and prkl-1 was further identified as playing a cell autonomous role. The origins of observed DD neuron anterior positioning defects were investigated during embryogenesis, in which 1.5 fold stage prkl-1(ok3182) embryos displayed delayed intercalation of the DD neurons. This represents a novel role for the PCP pathway in mediating DD neuronal intercalation.