The role of tinman, a mesodermal cell fate gene, in axon pathfinding during the development of the transverse nerve in Drosophila.

During the development of peripheral nerves, pioneer axons often navigate over mesodermal tissues. In this paper, we examine the role of the mesodermal cell determination gene tinman on cells that provide pathfinding cues in Drosophila. We focus on a subset of peripheral nerves, the transverse nerves, that innervate abdominal segments. During wildtype embryonic development, the transverse nerve efferents associate with glial cells located on the dorsal aspect of the CNS midline (transverse nerve exit glia). These glial cells have cytoplasmic extensions that prefigure the transverse nerve pathway from the CNS to the body wall musculature prior to transverse nerve formation. Transverse nerve efferents extend along this scaffold to the periphery, where they fasciculate with projections from a peripheral neuron--the LBD. In tinman mutants, the transverse nerve exit glia appear to be missing, and efferent fibers remain stalled at the CNS midline, without forming transverse nerves. In addition, fibers of the LBD neurons are often truncated. These results suggest that the lack of exit glia prevents normal transverse nerve pathfinding. Another prominent defect in tinman is the loss of all dorsal neurohemal organs, FMRFamide-expressing thoracic structures which likely contain the homologs of the transverse nerve exit glia in the thoracic segments. Our results support the hypothesis that the exit glia have a mesodermal origin and that glia play an essential role in determining transverse nerve axon pathways.

Dual muscarinic and nicotinic action on a motor program in Drosophila.

The effect of cholinergic agonists and antagonists on the central pattern generator of the pharyngeal muscles has been studied in third instar larvae of Drosophila. The pharyngeal muscles are a group of rhythmically active fibers involved in feeding. Bath application of the cholinergic agonists carbachol, muscarine, pilocarpine, and acetylcholine (ACh) to a semiintact preparation including the pharyngeal muscles and the central nervous system (CNS), initiated long-lasting endogenous-like bursting activity in the muscles. The muscarinic antagonists, atropine and scopolamine, blocked these responses as well as endogenous activity. Perfusion with nicotine elicited a short, tonic response that was marginally blocked by mecamylamine but not by curare, alpha-bungarotoxin, hexamethonium, or the muscarinic antagonists. This is the first time that a response to cholinergic drugs has been examined in Drosophila. The pharyngeal muscle preparation may prove to be a valuable system for studying mutations of cholinergic metabolism, receptors, and second messengers.

Single-channel K+ currents in Drosophila muscle and their pharmacological block.

Four types of nonvoltage-activated potassium channels in the body-wall muscles of Drosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA), Ba2+, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The KST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130 mM KCl solution), was blocked by TEA (Kd approximately 35 mM), and was the most sensitive to Ba2+ (complete block at 10(-4) M). A Ca(2+)-activated potassium channel, KCF.72 pS (130/130) mM KCl), was gated open at greater than 10(-8) m Ca2+, was the least sensitive to Ba2+ Kd of approximately 3 mM) and TEA (Kd of approximately 100 mM), and was not affected by quinidine. K2 was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1 mM. It also exhibited a half block at approximately 0.3 mM Ba2+ and approximately 25 mM TEA. A fourth channel type, K3, was the most sensitive to TEA (half block less than 1 mM). It displayed a partial block to Ba2+ at 10 mM, but no block by 0.1 mM quinidine. The blocking effects of TEA, Ba2+ and quinidine were reversible in all channels studied. The actions of TEA and Ba2+ appeared qualitatively different: in all four channels, TEA reduced the apparent unitary conductance, whereas Ba2+ decreased channel open probability.

Immunohistochemical localization of choline acetyltransferase during development and in Chats mutants of Drosophila melanogaster.

The distribution of choline acetyltransferase (CAT) in the nervous system of Drosophila melanogaster was determined by indirect immunohistochemical procedures using a monoclonal antibody specific to the enzyme. Immunoreactivity was first detected in the nervous system of 16 hr embryos, and increased considerably by the end of embryogenesis. Neuropil was preferentially stained, though cell bodies could also be observed. Staining was prominent in the CNS of all 3 larval instars but decreased substantially during the mid-pupal stage. Prior to eclosion, the level of immunoreactivity increased and the adult staining pattern became discernible. In the adult brain, staining was extensive, with numerous structures, such as the optic lobes and mushroom bodies, staining strongly. The adult thoracic ganglia were also moderately immunoreactive. These results imply a wide distribution of cholinergic neurons in the CNS of Drosophila. Immunoreactivity was also determined for 2 temperature-sensitive CAT mutants, Chats1 and Chats2. These files exhibit reduced CAT activity at permissive temperature, 18 degrees C, which eventually falls to undetectable levels after incubation at nonpermissive temperature, 30 degrees C. Chats2 mutants, after incubation at either 18 or 30 degrees C displayed virtually no staining. This result indicated that the immunoreactivity observed in wild-type flies was specifically associated with the enzyme encoded by the Cha gene. The intensity of staining in Chats1 mutants incubated at 18 degrees C appeared greater than in control flies, even though CAT enzyme activity in Chats1 is lower. This suggests that the enzyme molecule itself is structurally altered in Chats1 mutants. After incubation at 30 degrees C, staining in Chats1 flies decreased but did not disappear.