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Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na+/H+ exchanger 3 regulating factor (NHERF) family are conserved regulators of cortical specialization. ERM proteins function as membrane-actin linkers and as molecular scaffolds that organize the distribution of proteins at the membrane. NHERF proteins are PDZ-domain containing adapters that can bind to ERM proteins and extend their scaffolding capability. Here, we investigate how ERM and NHERF proteins function in regulating intestinal lumen formation in the nematode Caenorhabditis elegans. C. elegans has single ERM and NHERF family proteins, termed ERM-1 and NRFL-1, and ERM-1 was previously shown to be critical for intestinal lumen formation. Using CRISPR/Cas9-generated nrfl-1 alleles we demonstrate that NRFL-1 localizes at the intestinal microvilli, and that this localization is depended on an interaction with ERM-1. However, nrfl-1 loss of function mutants are viable and do not show defect in intestinal development. Interestingly, combining nrfl-1 loss with erm-1 mutants that either block or mimic phosphorylation of a regulatory C-terminal threonine causes severe defects in intestinal lumen formation. These defects are not observed in the phosphorylation mutants alone, and resemble the effects of strong erm-1 loss of function. The loss of NRFL-1 did not affect the localization or activity of ERM-1. Together, these data indicate that ERM-1 and NRFL-1 function together in intestinal lumen formation in C. elegans. We postulate that the functioning of ERM-1 in this tissue involves actin-binding activities that are regulated by the C-terminal threonine residue and the organization of apical domain composition through NRFL-1.

Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na

The apical domain of epithelial cells can acquire a diverse array of morphologies and functions, which is critical for the function of epithelial tissues. The Crumbs proteins are evolutionary conserved transmembrane proteins with essential roles in promoting apical domain formation in epithelial cells. The short intracellular tail of Crumbs proteins interacts with a variety of proteins, including the scaffolding protein Pals1 (protein associated with LIN7, Stardust in Drosophila). Pals1 in turn binds to a second scaffolding protein termed PATJ (Pals1-associated tight junction protein), to form the core Crumbs/Pals1/PATJ Crumbs complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and animal development. Moreover, the presence and functioning of orthologs of Pals1 and PATJ has not been investigated. Here, we identify MAGU-2 and MPZ-1 as the C. elegans orthologs of Pals1 and PATJ, respectively. We show that MAGU-2 interacts with all three Crumbs proteins as well as MPZ-1, and localizes to the apical membrane domain in a Crumbs-dependent fashion. Similar to crumbs mutants, a magu-2 null mutant shows no developmental or epithelial polarity defects. Finally, we show that overexpression of the Crumbs proteins EAT-20 or CRB-3 in the C. elegans intestine can lead to apical membrane expansion. Our results shed light into the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in theC. elegansintestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells, and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in theC. elegansintestine, bublin recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1. In mouse intestinal organoids, bublin localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.SummaryWe identify BBLN-1 as an evolutionary conserved regulator of lumen morphology in theC. elegansintestine. Loss ofbbln-1causes intermediate filament network reorganization that induces severe apical morphology defects. We also identify bublin (BBLN) as the mammalian ortholog, which can compensate for the loss of BBLN-1 inC. elegans.

During development, cell fate decisions are often highly stochastic, but with the frequency of the different possible fates tightly controlled. To understand how signaling networks control the cell fate frequency of such random decisions, we studied the stochastic decision of the Caenorhabditis elegans P3.p cell to either fuse to the hypodermis or assume vulva precursor cell fate. Using time-lapse microscopy to measure the single-cell dynamics of two key inhibitors of cell fusion, the Hox gene LIN-39 and Wnt signaling through the β-catenin BAR-1, we uncovered significant variability in the dynamics of LIN-39 and BAR-1 levels. Most strikingly, we observed that BAR-1 accumulated in a single, 1–4 ​h pulse at the time of the P3.p cell fate decision, with strong variability both in pulse slope and time of pulse onset. We found that the time of BAR-1 pulse onset was delayed relative to the time of cell fusion in mutants with low cell fusion frequency, linking BAR-1 pulse timing to cell fate outcome. Overall, a model emerged where animal-to-animal variability in LIN-39 levels and BAR-1 pulse dynamics biases cell fate by modulating their absolute level at the time cell fusion is induced. Our results highlight that timing of cell signaling dynamics, rather than its average level or amplitude, could play an instructive role in determining cell fate., Graphical abstract Image 1, Highlights • The fate of the C. elegans P3.p cell is stochastic. • β-catenin (BAR-1) accumulated in P3.p at the time of the cell fate decision. • There is variability in dynamics of Hox and β-catenin levels during the decision. • BAR-1 accumulated with variable pulse slope and time of pulse onset. • Pulse dynamics bias cell fate at the time of the cell fate decision.

Invertebrate and vertebrate nervous systems generate different types of dopaminergic neurons in distinct parts of the brain. We have taken a genetic approach to understand how the four functionally related, but lineally unrelated, classes of dopaminergic neurons of the nematode Caenorhabditis elegans, located in distinct parts of its nervous system, are specified. We have identified several genes involved in the generation of a specific dopaminergic neuron type that is generated from the so-called postdeirid lineage, called PDE. Apart from classic proneural genes and components of the mediator complex, we identified a novel, previously uncharacterized zinc finger transcription factor, ztf-6. Loss of ztf-6 has distinct effects in different dopamine neuron-producing neuronal lineages. In the postdeirid lineage, ztf-6 is required for proper cell division patterns and the proper distribution of a critical cell fate determinant, the POP-1/TCF-like transcription factor.

Cells in developing organisms must robustly assume the correct fate in order to fulfill their specific function. At the same time, cells are strongly affected by molecular fluctuations, i.e. ‘noise’, leading to inherent variability in individual cells. During development, some cells are thought to exploit such molecular noise to drive stochastic cell fate decisions, with cells randomly picking one cell fate out of several possible ones. Yet, how molecular noise drives such decisions is an open question. We address this question using a novel quantitative approach to study one of the best-understood stochastic cell fate decisions: the AC/VU decision inC. elegansgonad development. Here, two initially equivalent cells, Z1.ppp and Z4.aaa, interact, so that one cell becomes the anchor cell (AC) and the other a ventral uterine precursor cell (VU). It is thought that the symmetry is broken when small molecular fluctuations are amplified into cell fate by positive feedback loops in the Notch signaling pathway. To identify the noise sources that drive the AC/VU decision, we used a novel time-lapse microscopy approach to follow expression dynamics in live animals and single molecule FISH to quantify gene expression with single mRNA resolution. We found not only that random Z1.ppp/Z4.aaa birth order biased the decision outcome, with the first-born cell typically assuming VU fate, but that the strength of this bias and the speed of the decision decreased as the two cells were born closer together in time. Moreover, we find that the Notch ligandlag-2/Deltaexhibited strongly stochastic expression already in the two mother cells, Z1.pp/Z4.aa. Combining experiments with mathematical models, we showed that the resulting asymmetry inlag-2/Deltalevels inherited by the daughter cells, Z1.ppp/Z4.aaa, stochastic symmetry breaking when both cells are born at similar times. Together, our results suggest that two independent noise sources, birth order and stochasticlag-2/Deltaexpression, are exploited to amplify noise into cell fate in a manner that ensures a robust decision.

One challenge in modern medicine is to control epilepsies that do not respond to currently available medications. Since seizures consist of coordinated and high-frequency neural activity, our goal was to disrupt neurotransmission with a synaptic transmission mutant and evaluate its ability to suppress seizures. We found that the mutant shibire, encoding dynamin, suppresses seizure-like activity in multiple seizure–sensitive Drosophila genotypes, one of which resembles human intractable epilepsy in several aspects. Because of the requirement of dynamin in endocytosis, increased temperature in the shits1 mutant causes impairment of synaptic vesicle recycling and is associated with suppression of the seizure-like activity. Additionally, we identified the giant fiber neuron as critical in the seizure circuit and sufficient to suppress seizures. Overall, our results implicate mutant dynamin as an effective seizure suppressor, suggesting that targeting or limiting the availability of synaptic vesicles could be an effective and general method of controlling epilepsy disorders.

Genetic factors that influence seizure susceptibility can act transiently during the development of neural circuits or might be necessary for the proper functioning of existing circuits. We provide evidence that the Drosophila seizure-sensitive mutant easily shocked (eas) represents a neurological disorder in which abnormal functioning of existing neural circuits leads to seizure sensitivity. The eas(+) gene encodes for the protein Ethanolamine Kinase, involved in phospholipid biosynthesis. We show that induction of eas(+) in adult mutant flies rescues them from seizure sensitivity despite previously known developmental defects in brain morphology. Additionally, through cell-type-specific rescue, our results suggest a specific role for eas(+) in excitatory rather than inhibitory neural transmission. Overall, our findings emphasize an important role for proper phospholipid metabolism in normal brain function and suggest that certain classes of epilepsy syndromes could have the potential to be treated with gene therapy techniques.

SummaryHere, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (

© 2015 Elsevier Inc. This paper reviews Drosophila voltage-gated Na+channel mutations encoded by the para (paralytic) gene and their contributions to seizure disorders in the fly. Numerous mutations cause seizure-sensitivity, for example, parabss1, with phenotypes that resemble human intractable epilepsy in some aspects. Seizure phenotypes are also seen with human GEFS+ spectrum mutations that have been knocked into the Drosophila para gene, paraGEFS+and paraDSalleles. Other para mutations, paraST76and paraJSact as seizure-suppressor mutations reverting seizure phenotypes in other mutants. Seizure-like phenotypes are observed from mutations and other conditions that cause a persistent Na+current through either changes in mRNA splicing or protein structure.

SummaryWe report the larval CNS expression patterns for 6,650 GAL4 lines based on cis-regulatory regions (CRMs) from the Drosophila genome. Adult CNS expression patterns were previously reported for this collection, thereby providing a unique resource for determining the origins of adult cells. An illustrative example reveals the origin of the astrocyte-like glia of the ventral CNS. Besides larval neurons and glia, the larval CNS contains scattered lineages of immature, adult-specific neurons. Comparison of lineage expression within this large collection of CRMs provides insight into the codes used for designating neuronal types. The CRMs encode both dense and sparse patterns of lineage expression. There is little correlation between brain and thoracic lineages in patterns of sparse expression, but expression in the two regions is highly correlated in the dense mode. The optic lobes, by comparison, appear to use a different set of genetic instructions in their development.