Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior-posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.

During metazoan development, cell-fate diversity is brought about, in part, by asymmetric cell divisions. In Drosophila, bristle mechanosensory organs are composed of four different cells that originate from a single precursor cell, pI, after two rounds of asymmetric division. At each division, distinct fates are conferred on sister cells by the asymmetric segregation of Numb, a negative regulator of Notch signalling. Here we show that the orientation of the mitotic spindles and the localization of the Numb crescent follow a stereotyped pattern. Mitosis of pI is orientated parallel to the anteroposterior axis of the fly. We show that signalling mediated by the Frizzled receptor polarizes pI along this axis, thereby specifying the orientation of the mitotic spindle and positioning the Numb crescent. The mitoses of the two cells produced by mitosis of pI are orientated parallel and orthogonal, respectively, to the division axis of pI. This difference in cell-division orientation is largely independent of the identity of the secondary precursor cells, and is regulated by Frizzled-independent mechanisms.

Asymmetric distribution of fate determinants is a fundamental mechanism underlying the acquisition of distinct cell fates during asymmetric division. In Drosophila neuroblasts, the apical DmPar6/DaPKC complex inhibits Lethal giant larvae (Lgl) to promote the basal localization of fate determinants. In contrast, in the sensory precursor (pI) cells that divide asymmetrically with a planar polarity, Lgl inhibits Notch signaling in the anterior pI daughter cell, pIIb, by a yet-unknown mechanism. We show here that Lgl promotes the cortical recruitment of Partner of Numb (Pon) and regulates the asymmetric distribution of the fate determinants Numb and Neuralized during the pI cell division. Analysis of Pon-GFP and Histone2B-mRFP distribution in two-color movies confirmed that Lgl regulates Pon localization. Moreover, posterior DaPKC restricts Lgl function to the anterior cortex at mitosis. Thus, Lgl functions similarly in neuroblasts and in pI cells. We also show that Lgl promotes the acquisition of the pIIb cell fate by inhibiting the plasma membrane localization of Sanpodo and thereby preventing the activation of Notch signaling in the anterior pI daughter cell. Thus, Lgl regulates cell fate by controlling Pon cortical localization, asymmetric localization of Numb and Neuralized, and plasma-membrane localization of Sandopo.

SUMMARY1. The action of a short bath application of kainic acid (KA,(3)(4)(5) on the CA3 region of rat hippocampal slices has been studied with intracellular and extracellular recording techniques.2. KA evoked bursts which persisted for 10-15 min. In addition, after KA, electrical stimulation of various inputs to CA3 which elicited an EPSP-IPSP sequence in control conditions evoked an EPSP followed by a burst. This evoked response persisted for several hours after removal of KA suggesting the occurrence of a long-lasting modification of the synaptic properties of CA3 neurones.3. Intracellular recordings showed the spontaneous and evoked bursts to consist of five to ten action potentials riding on a depolarizing shift 10-25 mV in amplitude and 40-100 ms in duration. Both spontaneous and evoked bursts were followed by a long-lasting hyperpolarization 15-25 mV in amplitude and 1-15 s in duration.4. We propose that both spontaneous and evoked synchronized bursts are generated by a polysynaptic network since: (a) intracellularly recorded bursts were synchronized with the bursts in extracellular field recording; (b) bursts disappeared when synaptic transmission or Na+ action potential were blocked by cobalt (1 mm) or TTX (1 ,UM) respectively; (c) bursts were suppressed by elevated divalent cation concentration; (d) burst occurrence was independent of the membrane potential of the cell; (e) the depolarization shift that underlies the bursts was a linear function of the membrane potential and reversed in polarity at 0 mV. In addition, the evoked bursts were all-or-none events with a variable latency.5. Laminar profile analysis of the spontaneous and evoked bursts suggests that they were generated by synapses located on the distal apical segments of the dendrites of CA3 pyramidal cells.7. The persistence of the evoked bursts was neither due to a persistent change in cell excitability nor to a long-lasting reduction in GABAergic synaptic inhibition.8. Bath application of a high concentration of potassium (7 mm) also induced spontaneous and evoked bursts; the latter also persisted several hours after return to control medium.9. The N-methyl-D-aspartate (NMDA) antagonist, D-APV (D(-)-2-amino-5-phosphonovaleric acid) (30 /tM), did not block the spontaneous discharges induced by KA or high potassium, but prevented the long-lasting effects on the synaptic responses.10. In conclusion, we suggest that the long-lasting change of synaptic responses is Y. BEN-ARI AND M. GHO generated by the spontaneous synchronized discharges present during and shortly after the application of KA or high potassium. D-APV experiments suggest that NMDA receptors are involved in this change as in other forms of long-term synaptic plasticity.

Kinesin is believed to generate force for the movement of organelles in anterograde axonal transport. The identification of genes that encode kinesin-like proteins suggests that other motors may provide anterograde force instead of or in addition to kinesin. To gain insight into the specific functions of kinesin, the effects of mutations in the kinesin heavy chain gene (khc) on the physiology and ultrastructure of Drosophila larval neurons were studied. Mutations in khc impair both action potential propagation in axons and neurotransmitter release at nerve terminals but have no apparent effect on the concentration of synaptic vesicles in nerve terminal cytoplasm. Thus kinesin is required in vivo for normal neuronal function and may be active in the transport of ion channels and components of the synaptic release machinery to their appropriate cellular locations. Kinesin appears not to be required for the anterograde transport of synaptic vesicles or their components.Kinesin is a ubiquitous mechanochemical adenosine triphosphatase (ATPase) that can move toward the plus ends of microtubules in vitro (1, 2). The native molecule is a heterotetramer consisting of two identical heavy chains and two light chains (2). The kinesin heavy chain, and more specifically the NH 2 -terminal head of the heavy chain, appears to contain all of the mechanochemical elements necessary for generating microtubule-based movements (3). The light chains and the COOH-terminal tail of the heavy chain are thought to be involved in binding kinesin to vesicles or other cargo destined for microtubule-based transport (2, 4).On the basis of the polarity of kinesin's movement and its presence in neural cells, it was originally suggested that kinesin might act as a motor for anterograde axonal transport (1). It since has been shown that vesicle movement in axoplasm (both anterograde and retrograde) can be inhibited in vitro by an antibody that binds to the kinesin heavy chain (5). It also has been shown that kinesin heavy chain (khc) mutations in Drosophila cause behavioral defects that suggest a requirement for kinesin in normal neuromuscular functions (6). Although these data provide a persuasive argument that kinesin acts as a motor for axonal transport, the argument remains indirect.Kinesin is a member of a superfamily of kinesin-like proteins that share similar mechanochemical domains (7), and multiple members of the kinesin superfamily are co- We have taken advantage of khc mutations to study the effects of impaired kinesin function on specific physiological functions of motor neurons in Drosophila larvae. Action potential propagation by segmental nerves and synaptic transmission at neuromuscular junctions were tested in both the second (A2) and sixth (A6) abdominal segments of control and khc mutant larvae (9) . Two hypomorphic khc alleles (khc 5 and khc 6 ) were used to construct both control and mutant genotypes (10) because these alleles are severe enough to cause a strong behavioral phenotype in larvae (6), yet mild enough to per...