Copulation in C. elegans males requires a nuclear hormone receptor.

In Caenorhabditis elegans, uncoordinated (unc)-55 encodes a nuclear hormone receptor that is necessary for coordinated movement and male mating. An unc-55 reporter gene revealed a sexually dimorphic pattern: early in post-embryonic motor neurons in both sexes; and later in a subset of male-specific cells that included an interneuron and eight muscle cells. A behavioral analysis coupled with RNA interference (RNAi) revealed that males require UNC-55 to execute copulatory motor programs. Two mRNA isoforms (unc-55a and unc-55b) were detected throughout post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites. In unc-55 mutant males isoform a rescued the locomotion and mating defect, whereas isoform b rescued the mating defect only. Isoform b represents the first report of male-specific splicing in C. elegans. In addition, isoform b extended the number of days that transgenic unc-55 mutant males mated when compared to males rescued with isoform a, suggesting an anabolic role for the nuclear hormone receptor. The male-specific expression and splicing is part of a regulatory hierarchy that includes two key genes, male abnormal (mab)-5 and mab-9, required for the generation and differentiation of male-specific cells. We suggest that UNC-55 acts as an interface between genes involved in male tail pattern formation and those responsible for function.

Convergent genetic programs regulate similarities and differences between related motor neuron classes in Caenorhabditis elegans.

How do genetic programs create features common to a specific cell or tissue type while generating modifications necessary for functional diversification? We have addressed this question using the nematode Caenorhabditis elegans. The dorsal D (DD) and ventral D (VD) motorneurons (mns), referred to collectively as the D mns, compose a cross-inhibitory network that contributes to the animal's sinuous locomotion. The D mns share a number of structural and functional features, but are distinguished from one another by their synaptic patterns and the expression of a neuropeptide gene. Our findings suggest that the similarities and differences are generated at the transcriptional level. UNC-30 contains a homeodomain and activates structural and functional genes expressed in both classes. UNC-55 is a nuclear receptor expressed in the VD mns that is necessary for generating features that distinguish the two classes of D mns from one another. In unc-55 mutants, the VD mns adopt the DD mn synaptic pattern and peptide expression profile. Conversely, ectopic expression of unc-55 in the DD mns causes them to adopt VD mn features. The promoter of the neuropeptide gene expressed in the DD mns contains putative binding sites for both UNC-30 and UNC-55; alteration of these sites suggests that UNC-55 represses the ability of UNC-30 to activate a subset of genes that are expressed in the DD mns but not in the VD mns. Thus UNC-55 acts as a switch for the features that distinguish these two functionally related classes of mns.

A CUB-serine protease in the olfactory organ of the spiny lobster Panulirus argus.

csp, a gene encoding a protein with high sequence identity to trypsinlike serine protease and CUB domains, was identified from a cDNA library from the olfactory organ (antennular lateral flagellum) of the spiny lobster Panulirus argus. The full-length cDNA sequence of csp is 1801 bp, encoding a protein of 50.25 kD, with three domains: signal peptide, trypsinlike serine protease, and CUB (named for a class of compounds including Complement subcomponents Clr/Cls, Uegf, and Bone morphogenic protein-1). RT-PCR, Northern blots, and immunoblots showed that csp is predominantly expressed in the lateral flagellum and eyestalk. Immunocytochemistry showed that Csp is present in olfactory (aesthetasc) sensilla around auxiliary cells (glia that surround the inner dendrites of olfactory receptor neurons, ORNs) and ORN outer dendrites. We propose that Csp is expressed and secreted by auxiliary cells, associates with ORN cell membranes or extracellular matrix via the CUB domain, and has trypsinlike activity. In the eyestalk, Csp is associated with cells surrounding axons between neuropils of the eyestalk ganglia. Possible functions in the olfactory organ and eyestalk are discussed. To our knowledge, this is the first report from any olfactory system of a gene encoding a protein with serine protease and CUB domains.

UNC-55, an orphan nuclear hormone receptor, orchestrates synaptic specificity among two classes of motor neurons in Caenorhabditis elegans.

Loss of UNC-55 function in the nematode Caenorhabditis elegans causes one motor neuron class, the ventral D (VD) motor neurons, to adopt the synaptic pattern of another motor neuron class, the dorsal D (DD) motor neurons. Here we show that unc-55 encodes a member of the nuclear hormone receptor gene family that is similar to the vertebrate chicken ovalbumin upstream promoter transcription factors. Although the VD and DD motor neuron classes arise from different lineages at different developmental stages, they share a number of structural and functional features that appear to be the product of identical genetic programs. UNC-55 is expressed in the VD but not the DD motor neurons to modify this genetic program and to create the synaptic pattern that distinguishes the two motor neuron classes from one another.

Identification and partial characterization of putative taurine receptor proteins from the olfactory organ of the spiny lobster.

To explore the initial stages of olfactory transduction, we have used biochemical techniques to characterize proteins associated with the dendritic plasma membrane from the olfactory receptor neurons of the spiny lobster Panulirus argus. In particular, we have studied proteins that interact with taurine, an amino acid that is an important odorant for this species. The cross-linker bis(sulfosuccinimidyl)suberate (BS3) was used to covalently link [3H]-taurine to cell surface proteins on membrane from the aesthetasc (olfactory) sensilla of the lateral filament of the antennule. A radioligand-receptor binding assay was used to show that this cross-linkage was highly specific for taurine at 0.2 mM BS3. In inhibition studies, of all the unlabeled odorants tested at excess concentrations (taurine, L-glutamate, adenosine-5'-monophosphate), only taurine significantly inhibited the cross-linkage of [3H]-taurine to the membrane. Membranes containing cross-linked proteins were solubilized, and proteins were separated on SDS-PAGE and examined with autoradiography. Bands with molecular weights of 100, 82, 62, 51, and 34kD were evident on the gels. However, only the 100 and 62 kD bands were consistently labeled with [3H]-taurine, and this labeling was completely inhibited in the presence of excess unlabeled taurine but not adenosine-5'monophosphate. The taurine-evoked behavioral search response of spiny lobsters was significantly reduced following treatment of their antennules with BS3 + taurine as compared with animals treated with BS3 alone, suggesting that the taurine-labeled binding proteins include taurine receptor proteins involved in the first stage of olfactory transduction.

Dynamic interactions between nerve and muscle in Caenorhabditis elegans.

Synaptogenesis among developing motoneurons and muscles was examined in the nematode Caenorhabditis elegans. In this animal embryonic precursor cells give rise to regionally localized, contiguous clones of muscle cells that form two dorsal and two ventral sets that run longitudinally along the body wall. Ablation of selected embryonic muscle precursors resulted in gaps in the posterior dorsal muscle quadrants. We compared the morphological development of GABAergic locomotory neurons in the presence and absence of their target muscle cells. The results led to four main conclusions: (1) target muscle cells are not required for the morphological differentiation of the motoneurons; (2) target muscle cells appear to be required for the formation of presynaptic varicosities by the motoneurons; (3) embryonic muscle cells serve as a guide for migrating postembryonic muscle cells and in the absence of these guides the postembryonic muscles often assume ectopic locations; and (4) in the presence of ectopic muscle cells, the GABAergic locomotory neurons sprouted and formed branches that contributed to ectopic neuromuscular junctions.

Genetic transformation of the synaptic pattern of a motoneuron class in Caenorhabditis elegans.

Caenorhabditis elegans possesses two classes of inhibitory locomotory neurons, the DD and VD motoneurons (mns), and they form complementary components of a cross-inhibitory neuronal network innervating dorsal and ventral body muscles. The DD and VD mns (collectively called the D mns) share a number of morphological and neurochemical features, and mutations in a number of different genes disrupt both cell types in identical ways; however, the DD and VD mns have different lineal origins and different synaptic patterns. Given the number of phenotypic features shared by the D mns, it was of interest to determine what is responsible for the synaptic patterns that distinguish them. An analysis of the locomotory defect along with a genetic epistasis test suggested that unc-55 mutations alter the function of the VD but not the DD mns. Correlated with the defective locomotory behavior of unc-55 mutants was an alteration in the distribution of varicosities, structures associated with presynaptic elements, on the VD mns. The pattern of varicosities of the unc-55 VD mns resembled that of the wild-type DD mns. Moreover, the selective removal of the DD mns revealed that unc-55 VD mns had adopted a functional role appropriate for the DD mns. Thus, unc-55 appears to be involved in producing the synaptic patterns that distinguish the two D mn classes from one another; when the gene is mutated the VD and DD mns become structurally similar and functionally equivalent.

Repeating patterns of motoneurons in nematodes: the origin of segmentation?

Evolutionarily diverse groups of animals share numerous similarities as individual neurons are assembled into functional neural circuits. One example is the hierarchical sequence of events that individual nerve cells follow during morphological development. In the initial step a presumptive neuron is generated and positioned appropriately. Second, the undifferentiated cell elaborates a growth cone capable of interacting with extrinsic cues and leading the presumptive axonal process as it is guided into areas where potential synaptic targets reside. Finally, the differentiating nerve cell selects among appropriate and inappropriate target cells as it completes the process of selective synaptogenesis. The extracellular matrix molecule laminin provides a second example, this time at the molecular level. Biochemical and genetic studies have shown that this molecule directs process guidance of neurons in vertebrates, annelids, and nematodes. In both examples an interest in neural development has provided a window through which evolutionarily processes have been revealed. The free-living soil nematode Caenorhabditis elegans possesses several features that collectively place it in a rather unique position among metazoans and has allowed genetic and cellular studies to be integrated at the level of identified neurons and neural circuits. This review will focus on developmental studies of C. elegans locomotory neural circuits. General issues that will be addressed are the similarities and differences among different taxa regarding: the relationship between cell lineage and cell fate determination in generating reiterative neural patterns; pioneer cells and the molecular basis for process guidance and finally genetic epigenetic events involved in sculpting highly specific synaptic patterns.

Changing synaptic specificities in the nervous system of Caenorhabditis elegans: differentiation of the DD motoneurons.

During postembryonic development, the DD motoneurons in the nematode Caenorhabditis elegans completely reorganize their pattern of synapses. Ablation of a pair of embryonic precursors results in the absence of this entire class of motoneurons. In their absence animals exhibit two developmentally distinct locomotory defects. The transition period from one defect to the other is correlated with the synaptic reorganization of the DD mns. Mutations in a gene (unc-123) have been isolated that exhibit locomotory defects similar to those of the ablated adult animals. Genetic and cellular analyses of one of these alleles suggest that the unc-123 gene product may be involved in the reestablishment of functional synapses in these neurons.

Metamorphic-like changes in the nervous system of the nematode Caenorhabditis elegans.

During postembryonic development of the nematode Caenorhabditis elegans, one class of embryonic motoneurons, the DD cells, respecifies its pattern of synaptic connections. At the same time, a closely related set of postembryonic motoneurons, the VD cells, complete differentiation and assume a pattern of connections equivalent to the original pattern of the DD cells. These types of changes are reminiscent of changes observed in the nervous systems of animals as they undergo metamorphosis. The DD and VD neurons arise through different lineage mechanisms and in the adult, receive different synaptic inputs and make different outputs. The embryonic DD motoneurons are clonally related to one another; whereas the postembryonic VD motoneurons are produced by a repeated sublineage in which each stem cell generates four or five cell types in addition to the VD cells. In spite of these differences, it has been possible to identify only one gene by mutation that effects one of the two motoneuronal classes. Mutations in the gene unc-55 (unc meaning uncoordinated) cause the VD cells to become essentially identical to the DD cells; thus the unc-55 gene product appears necessary and sufficient to transform homeotically the pattern of synaptic connections of an entire class of motoneuron.

Cell-cell interactions in the guidance of late-developing neurons in Caenorhabditis elegans.

The initial outgrowth of developing neuronal processes can be affected by a number of extrinsic interactions. Cell-cell interactions are also important in a later stage of neuronal outgrowth when processes grow into the region of their targets. The correct positioning of the process of a postembryonic sensory neuron, the touch cell AVM of the nematode Caenorhabditis elegans, at its synaptic targets requires the presence of a pair of embryonic interneurons, the BDU cells. These cells receive synapses from AVM but do not participate in the touch reflex circuit. Therefore, the AVM-BDU synapses may be required to stabilize the association between these cells and assist in the guidance of the AVM processes through a mature neuropil.

Rules for neural development revealed by chimaeric sensory systems in crickets.

Many sensory systems are organized so that the afferent projection forms a topographic map of the sensory surface within the central nervous system (CNS). The information necessary to create such a map may be available to the neuronal cell body based on its position in the receptor array, and this 'positional information' is translated into an axonal arborization in the proper part of the CNS. To study how the location of a cell body within the sensory surface determines the termination pattern of its axon within the CNS, we have transplanted epidermis, containing identified sensory neurones, from a black cricket to a tan cricket. As we report here, when epidermis is transplanted to an unusual location in the receptor array, newly generated neurones are produced along the borders of the graft. These neurones arborize in locations that are appropriate neither to their new position nor to their original position in the array, but rather to a position somewhere in between. This is direct evidence for the idea that positional information guides the differentiation and ultimately the synaptic connections of insect sensory neurones.

Neurospecificity in the cricket cercal system.

Studies of neurospecificity in the cricket cercal sensory system are reviewed and a decade of experimentation is examined in the light of recently obtained anatomical data. The nearly complete description of the anatomy indicates that the excitatory receptive fields of directionally-selective interneurones are a joint function of an orderly afferent projection and the dendritic structure of the first order interneurones. The detailed understanding of the anatomy is shown to be a powerful tool in the interpretation of previously published physiological experiments and the design of new ones. The mechanisms which shape the orderly afferent projection are then described and compared with the work on vertebrate sensory systems. It is concluded that both positional interactions of the type conceived by Sperry (1963) and competitive interactions of the type conceived by Hubel, Wiesel & LeVay (1977) are involved in producing the cercal afferent projection. Thus the two main components of the neurospecificity concept are shown to exist in the cricket nervous system. The limits of a purely anatomical approach to the study of neurospecificity are considered in light of the work on this cricket sensory system.

Chemical excitation of Limulus photoreceptors. II. Vanadate, GTP-gamma-S, and fluoride prolong excitation evoked by dim flashes of light.

We treated Limulus ventral photoreceptors with the phosphatase inhibitors fluoride, vanadate, and GTP-gamma-S [guanosine-5'0-(3-thiotriphosphate)] under various conditions of illumination and external calcium concentrations. In the dark in low-calcium (1 mM) artificial seawater (ASW), fluoride-induced discrete waves cluster together in time. Under these conditions, the intervals between waves were found to be correlated, and there were excess short intervals beyond the number expected from an exponential interval distribution. To assess the effects of the inhibitors on the light response, we stimulated ventral receptors with a series of dim flashes and averaged the current response under voltage clamp. In ASW, vanadate and GTP-gamma-S prolong the decay of the averaged response to dim test flashes, but prolongation does not always accompany the induction of discrete waves in the dark. Prolongation induced by vanadate in normal-calcium (10 mM) ASW was enhanced in low-calcium (1 mM Ca2+) ASW. Many individual response records suggest that prolongation results from extra discrete waves late in the light response, whereas others reveal long-lasting complex waveforms that cannot easily be resolved into discrete waves. The apparent effect of the inhibitors on the light response is to allow a single photoactivated rhodopsin molecule to produce multiple discrete waves and complex long-lasting events. We suggest that both prolongation of the light response and clustering of waves in the dark result from inhibition of a step in the pathway of visual transduction, in which GTP hydrolysis normally helps to turn off the production of both light-evoked and spontaneous waves.

Giant interneurons mediating equilibrium reception in an insect.

In the burrowing cockroach Arenivaga, two giant interneurons in each connective of the ventral nerve cord provide gravity orientation information. The interneurons receive input from plumb bob-like equilibrium receptors on the ventral surface of the cerci. Ouir results support the theory that the cerci of cockroaches are specialized equilibrium organs.