Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.

Presynaptic glutamic acid decarboxylase is required for induction of the postsynaptic receptor field at a glutamatergic synapse.

We have systematically screened EMS-mutagenized Drosophila for embryonic lethal strains with defects in glutamatergic synaptic transmission. Surprisingly, this screen led to the identification of several alleles with missense mutations in highly conserved regions of Dgad1. Analysis of these gad mutants reveals that they are paralyzed owing to defects in glutamatergic transmission at the neuromuscular junction. Further electrophysiological and immunohistochemical examination reveals that these mutants have greatly reduced numbers of postsynaptic glutamate receptors in an otherwise morphologically normal synapse. By overexpressing wild-type Dgad1 in selected neurons, we show that GAD is specifically required in the presynaptic neuron to induce a postsynaptic glutamate receptor field, and that the level of postsynaptic receptors is closely dependent on presynaptic GAD function. These data demonstrate that GAD plays an unexpected role in glutamatergic synaptogenesis.

An extraretinally expressed insect cryptochrome with similarity to the blue light photoreceptors of mammals and plants.

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate sensitivity to blue light or longer wavelengths, respectively. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. We now have identified and characterized a candidate blue light photoreceptor gene in Drosophila (DCry) that is homologous to the cryptochrome (Cry) genes of mammals and plants. The DCry gene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded by DCry is approximately 50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment, DCry mRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissue in situ hybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with the Drosophila clock neurons. Interestingly, DCry mRNA abundance oscillates in a circadian manner in Drosophila head RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouse Cry1 mRNA, which is expressed in clock tissues. Finally, we show that changes in DCry gene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity.