Altered LARK expression perturbs development and physiology of the Drosophila PDF clock neurons
Introduction
Post-transcriptional regulatory events are essential for the function of the molecular oscillator governing circadian behavior. These include post-translational modifications by kinases, phosphatases and other regulatory factors that modulate clock protein intracellular localization, stability and activities (Young and Kay, 2001, Lee et al., 2003b, Harms et al., 2004, Liu, 2005, Vanselow and Kramer, 2007, Zheng and Sehgal, 2008). In addition, it has become apparent that circadianly regulated RNA-binding proteins (RBPs) have important post-transcriptional circadian functions in several different species (Heintzen et al., 1997, McNeil et al., 1998, Dockendorff et al., 2002, Morales et al., 2002, Liu, 2005, Iliev et al., 2006, Garbarino-Pico and Green, 2007). The Drosophila LARK RBP and its mammalian homolog mLARK (a.k.a. RBM4) display circadian changes in abundance and mediate post-transcriptional events that are critical for circadian timing in flies and mammals, respectively (Newby and Jackson, 1993, Newby and Jackson, 1996, McNeil et al., 1998, Zhang et al., 2000, Schroeder et al., 2003, Kojima et al., 2007). Our recent studies demonstrate that fly LARK is associated with a large number of mRNA target molecules, in vivo, and suggest that the RBP regulates certain targets by interacting with the microRNA (miRNA) pathway (Huang et al., 2007); mouse LARK has also been implicated in miRNA-mediated gene regulation (Hock et al., 2007). Surprisingly, one recent study demonstrates in vivo interactions of LARK with the Fragile X Mental Retardation Protein (FMRP) – another RBP implicated in microRNA pathway function – suggesting that the two RBPs might cooperate to regulate certain target RNAs (Sofola et al., 2008). LARK target mRNAs encode proteins important for many developmental and physiological processes including neuronal survival, neurite growth and pathfinding, neuronal excitability, synaptic function, circadian biology and others. Although the circadian role of LARK is well established (Newby and Jackson, 1993, Newby and Jackson, 1996, McNeil et al., 1998, Zhang et al., 2000, McNeil et al., 2001, Schroeder et al., 2003), a mechanistic understanding of this function remains a goal of current work. Furthermore, despite the identification of potential LARK targets that function in neuronal growth and differentiation (Huang et al., 2007), the role of LARK in neural development has not been studied.
In this report, we have chosen a specific group of neurons in the Drosophila brain known as the Pigment-Dispersing Factor (PDF)-expressing ventral lateral neurons (PDF-positive LNvs, hereafter referred to as “PDF neurons”) as a model to study the diverse functions of LARK. The PDF neurons were chosen for several reasons. They represent a small group of neurons with well characterized anatomy (Helfrich-Forster, 1997, Helfrich-Forster, 2003, Helfrich-Forster et al., 2007). In addition, the LNvs are known to be critical components of the circadian circuitry regulating locomotor activity. LARK overexpression in the LNvs was previously shown to cause arrhythmic locomotor activity, but it was not determined whether this phenotype was a consequence of altered clock neuron physiology or development (Schroeder et al., 2003). Both developmental and physiological alterations of the LNvs have dramatic effects on circadian function. Previous studies have shown that genetic ablation of the PDF neurons causes defects in circadian locomotor activity (Renn et al., 1999). Furthermore, a number of manipulations that alter the physiology of the LNvs, including the elimination of PDF neuropeptide (Renn et al., 1999), overexpression of the PER clock protein (Blanchardon et al., 2001), blockade of synaptic transmission by expression of tetanus toxin (Blanchardon et al., 2001), and modification of neuronal membrane excitability (Nitabach et al., 2002, Nitabach et al., 2006, Wu et al., 2008), all dramatically affect the circadian control of locomotor activity. Here we show that LARK overexpression affects development of the PDF neurons. Surprisingly, however, increased LARK expression exclusively at the adult stage abolishes rhythmic locomotor activity without affecting neuronal morphology, suggesting a dual role for LARK in development and physiology of the LNvs. In addition, we show that the effect of LARK overexpression on two key components of the molecular clock, PERIOD (PER) and PAR Domain Protein1ɛ (PDP1ɛ), resembles that caused by electrical silencing of the PDF neurons. To determine if LARK might regulate neuronal activity, we recorded from the larval neuromuscular junctions (NMJs) of lark null and overexpression mutants; those studies indicated that changes in the abundance of this RBP can regulate excitability. Taken together with previous findings that LARK is associated with mRNAs encoding potassium channels, an intriguing model is that an increased amount of the RBP promotes expression of these channels, resulting in decreased PDF cell membrane excitability and abolition of the circadian locomotor activity rhythm.
Section snippets
Dosage-dependent effects of LARK expression on development of the PDF-expressing small lateral neurons (s-LNvs)
Overexpression of LARK in the PDF neurons was previously found to cause behavioral arrhythmicity without altering survival of the neurons or the gross morphology of neuronal projections (Schroeder et al., 2003). Those studies, however, utilized flies carrying only single copies of the pdf-gal4 driver and uas-lark transgenes. In our recent studies, we found that increasing the dosage of either the pdf-gal4 driver or uas-lark responder transgene caused obvious developmental defects. For example,
Diverse roles of LARK in neural development and physiology
Post-transcriptional regulation of gene expression, via RNA-binding proteins (RBPs), is a hallmark of nervous system development and function (Robinow and White, 1991, Antic et al., 1999, Keene and Lager, 2005, Keene, 2007, Thyagarajan et al., 2007, Zearfoss et al., 2008). For example, the HuD RBP regulates neuronal differentiation, maintenance and plasticity (reviewed in (Deschenes-Furry et al., 2006)). Another RBP, the Fragile X mental retardation protein, FMRP, is critical for synapse
Fly strains
The pdf-gal4 lines were provided by Jae Park (University of Tennessee, Knoxville, TN). w1118, UAS-mCD8GFP , and ts-tubulin-gal80 were obtained from the Bloomington Stock Center. UAS-lark (23A) was described previously (Schroeder et al., 2003). All GAL4, UAS, and GAL80 transgenes were crossed onto the w1118 genetic background. Flies were reared on a modified cornmeal/agar medium (medium1 of Newby and Jackson, 1991) in an 12:12 light–dark cycle at 23 °C with constant humidity (∼ 60%) unless
Acknowledgments
We thank Jae Park for providing the pdf-gal4 lines, Dr. Lai Ding and Dr. Alenka Lovy-Wheeler (Tufts Imaging Facility) for assistance with confocal imaging, and Ralf Stanewsky, Paul Hardin, K. Ranga Rao, and Gerry McNeil for providing the anti-PER, anti-PDP1ɛ, anti-PDH and anti-LARK antibodies, respectively. This research was supported by NIH R01 HL59873 (FRJ), Department of Defense W81XWH-04-1-0272 (MS), and center grant NIH P30 NS047243 to the Tufts Center for Neuroscience Research (PI, FRJ).
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