Abstract
BackgroundWhile developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties.ResultsTo test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons we utilized the molecular genetics of Drosophila to manipulate the expression of Eve in identified motoneurons. We show that increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, we observed a decrease in both the fast K+ conductance (IKfast) and amplitude of quantal cholinergic synaptic input. We used charybdotoxin to pharmacologically separate the individual components of IKfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca2+-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRα-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRα-96Aa.ConclusionTaken together, our findings demonstrate, for the first time, that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. Our data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties.
Highlights
While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons
Given that motoneuron axon guidance is under combinatorial control of a set of transcription factors, this study addresses whether the acquisition of electrical properties, at least in early development, might be influenced by these same regulatory proteins
Our choice of motoneuron was guided by the fact that aCC/RP2 endogenously express eve [18] and manipulation of its expression should, result in altered electrophysiology if this transcription factor contributes to setting electrical properties in these neurons
Summary
While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Recent modeling studies indicate that extrinsic factors predominate given that indistinguishable network activity can arise from widely disparate combinations of individual conductances [8]. These analyses are based on neurons that are already components of functional networks. In the early stages of their development, embryonic neurons are largely deprived of extrinsic factors such as synaptic activity and sensory feedback It is in this relatively early phase of electrical development that genetic determination will likely predominate to ensure, at a minimum, the expression of a default set of ion channel proteins. After a neuron has the capability to contribute to a network can other, largely extrinsic factors, begin to shape the final mix of ion channels expressed
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