Neurohemal Organ Research Articles

Pigment-dispersing hormone in Daphnia interneurons, one type homologous to insect clock neurons displaying circadian rhythmicity.

We report identification of a beta-type pigment-dispersing hormone (PDH) identical in two water flea species, Daphnia magna and Daphnia pulex. It has been identified by cloning of precursors, chromatographic isolation from tissue extracts followed by immunoassays and de novo-mass spectrometric sequencing. The peptide is restricted to a complex system of distinct interneurons in the brain and visual ganglia, but does not occur in neurosecretory cells projecting to neurohemal organs as in decapod crustaceans. Thirteen neuron types individually identified and reconstructed by immunohistochemistry were almost identical in terms of positions and projection patterns in both species. Several neurons invade and form plexuses in visual ganglia and major brain neuropils including the central body. Five neuron types show contralateral pathways and form plexuses in the lateral, dorsal, or postlateral brain neuropils. Others are local interneurons, and a tritocerebral neuron connects the protocerebrum with the neuropil of the locomotory second antenna. Two visual ganglia neuron types lateral to the medulla closely resemble insect medulla lateral circadian clock neurons containing pigment-dispersing factor based upon positional and projectional criteria. Experiments under 12:12h light/dark cycles and constant light or darkness conditions showed significant circadian changes in numbers and activities of one type of medulla lateral PDH neuron with an acrophase in the evening. This simple PDH system shows striking homologies to PDH systems in decapod crustaceans and well-known clock neurons in several insects, which suggests evolutionary conservation of an ancient peptidergic interneuronal system that is part of biological clocks.

Deficiency of prohormone convertase dPC2 (AMONTILLADO) results in impaired production of bioactive neuropeptide hormones in Drosophila

Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.

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.

Enhancement of Muscle Contraction in the Stomach of the Crab Cancer borealis: a Possible Hormonal Role for GABA

Gamma-aminobutyric acid (GABA) is best known as an inhibitory neurotransmitter in the mammalian central nervous system. Here we show, however, that GABA has an excitatory effect on nerve-evoked contractions and on excitatory junctional potentials (EJPs) of the gastric mill 4 (gm4) muscle from the stomach of the crab Cancer borealis. The threshold concentration for these effects was between 1 and 10 micromol l(-1). Using immunohistochemical techniques, we found that GABA is colocalized with the vesicle-associated protein synapsin in nearby nerves and hence is presumably released there. However, since these nerves do not innervate the muscle directly, we conclude that these release sites are not the likely source of the GABA responsible for muscle modulation. We also extracted hemolymph from the crab pericardial cavity, which contains the pericardial organs, a major neurosecretory structure. Through reversed-phase liquid chromatography-mass spectrometry analysis we determined the concentration of GABA in the hemolymph to be 3.3 +/- 0.7 micromol l(-1), high enough to modulate the muscle. These findings suggest that the gm4 muscle could be modulated by GABA produced by and released from a distant neurohemal organ.

Ultrastructural and neurochemical architecture of the pituitary neural lobe of Xenopus laevis

The melanotrope cell in the amphibian pituitary pars intermedia is a model to study fundamental aspects of neuroendocrine integration. They release α-melanophore-stimulating hormone (αMSH), under the control of a large number of neurochemical signals derived from various brain centers. In Xenopus laevis, most of these signals are produced in the hypothalamic magnocellular nucleus (Mg) and are probably released from neurohemal axon terminals in the pituitary neural lobe, to stimulate αMSH-release, causing skin darkening. The presence in the neural lobe of at least eight stimulatory factors implicated in melanotrope cell control has led us to investigate the ultrastructural architecture of this neurohemal organ, with particular attention to the diversity of neurohemal axon terminals and their neurochemical contents. Using regular electron microscopy, we here distinguish six types of neurohemal axon terminal, on the basis of the size, shape and electron-density of their secretory granule contents. Subsequently, we have identified the neurochemical contents of these terminal types by immuno-electron microscopy and antisera raised against not only the ‘classical’ neurohormones vasotocin and mesotocin but also brain-derived neurotrophic factor, cocaine- and amphetamine-regulated transcript peptide, corticotropin-releasing factor, metenkephalin, pituitary adenylyl cyclase-activating polypeptide, thyrotropin-releasing hormone and urocortin-1. This has revealed that each terminal type possesses a unique set of neurochemical messengers, containing at least four, but in some cases up to eight messengers. These results reveal the potential of the Mg/neural lobe system to release a wide variety of neurochemical messengers in a partly co-ordinated and partly differential way to control melanotrope cell activity as well as ion and water balance regulatory organs, in response to various, continuously changing, environmental stimuli.

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly, Protophormia terraenovae

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum-hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg(7)]-corazonin and [Arg(7)]-corazonin(3)(-)(11)) in the PL, 13 peptides (including myosuppressin, [Arg(7)]-corazonin and [Arg(7)]-corazonin(3-11)) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 degrees C) and diapause-averting conditions (LD 18:6 at 25 degrees C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd-10th amino acids were sequenced as -YRKPPFNGS-, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.

Sex pheromone composition of the cotton caterpillar, Palpita indica (Lepidoptera: Pyralidae), in Korea

This study was conducted to investigate the sex pheromone composition of Palpita (= Diaphania) indica (Saunders) (Lepidoptera: Pyralidae) in Korea. Two sex pheromone components, E11-16:Al and E,E-10,12-16:Al, were identified by GC and GC-EAD analysis of sex pheromone gland extracts. The mean ratios of the two components, E11-16:Al and E,E-10,12-16:Al, were about 6.5:3.5 and 7.5:2.5 in gland extracts and in SPME collection, respectively. In field bioassays, maximum attraction occurred in traps baited with a 7:3 ratio of E11-16:Al and E,E-10,12-16:Al. The head extracts of P. indica stimulated the sex pheromone production of P. indica, as well as Hez-PBAN and PssPT, indicating that a PBAN-like substance exists in the head extracts of P. indica. Whole-mount immunocytochemistry showed that three groups of neurosecretory cells showed PBAN-like immunoreactivity in the SEG of P. indica. The PBAN-like immunoreactivity connected to the Corpora Cardiaca, a neurohemal organ. Also, PBAN-like immunoreactivity was found in the brain and in the thoracic and third/fourth abdominal ganglia. The addition of sex pheromone components of Peridroma saucia to the sex pheromone of P. indica greatly improved the attraction of P. indica males. The mixing of the sex pheromone components of S. exigua did not significantly increase the catches of P. indica, while the sex pheromone of H. armigera completely inhibited the attractiveness.

Peptidomic survey of the locust neuroendocrine system

Neuropeptides are important controlling agents in animal physiology. In order to understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. We here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, we analysed the brain, suboesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e. pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosaemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.

Novel gene encoding precursor protein consisting of possible several neuropeptides expressed in brain and frontal ganglion of the silkworm, Bombyx mori

A novel gene ( BmK5) expressed in the central nervous system of the silkworm, Bombyx mori, was isolated using a cDNA subtraction method. BmK5 was first cloned as a candidate regulator of diapause hormone release from subesophageal ganglion via corpus cardiacum–corpus allatum into the hemolymph; however, subsequent analyses revealed that the gene expression patterns in brain–subesophageal ganglion complexes did not differ between diapause and nondiapause egg producers. The deduced amino acid sequence showed the characteristics of secretory protein precursor or nuclear localization protein. Immunohistochemical experiments with an anti-BmK5 antibody revealed that BmK5 precursor protein exists in the cytoplasm of specific cells of brain and frontal ganglion, but not in the nuclei. In addition, a peptide (GSGTKVGGAGAATKVVTKSGS-NH 2) possibly processed from the BmK5 precursor protein was immunohistochemically detected in the axons connecting the anti-BmK5 antibody-positive cells to the neurohemal organ, corpus cardiacum–corpus allatum. These results suggest that BmK5 encodes a precursor of the novel neurosecretory protein and that several mature peptides are released into the hemolymph via the corpus cardiacum–corpus allatum, although the functions of these peptides are yet unclear.

Neuropeptides in Heteroptera: Identification of allatotropin-related peptide and tachykinin-related peptides using MALDI-TOF mass spectrometry

Recently, the peptidomic analysis of neuropeptides from the retrocerebral complex and abdominal perisympathetic organs of polyphagous stinkbugs (Pentatomidae) revealed the group-specific sequences of pyrokinins, CAPA peptides (CAPA-periviscerokinins/PVKs and CAPA-pyrokinin), myosuppressin, corazonin, adipokinetic hormone, and short neuropeptide F. In this study, we used mass spectrometric profiling of nervous tissue from the species-rich taxon Hemiptera to identify products of two previously unobserved neuropeptide genes from these species, namely allatotropin-related peptide (ATRP) and tachykinin-related peptides (TKRPs). Since neither TKRPs nor allatotropin are accumulated in neurohemal organs, immunocytochemical data were analyzed to find potential accumulation sites within the central nervous system. By mass spectrometry, TKRPs were found to be accumulated in the antennal lobes, and ATRP was identified in the most posterior region of the abdominal ventral nerve cord and fourth abdominal nerves. In addition to neuropeptides from stink bugs, TKRPs and ATRP were also identified from the distantly related bugs Oncopeltus fasciatus (Lygaeidae) and Pyrrhocoris apterus (Pyrrhocoridae). In total, six TKRPs and one ATRP from each species could be elucidated by tandem mass spectrometry. The ATRP of all species is sequence-identical with Locusta migratoria accessory gland myotropin-1 (Lom-AG-MT-1), a member of the highly conserved insect allatotropin family.

A newly identified extrinsic input triggers a distinct gastric mill rhythmviaactivation of modulatory projection neurons

Neuronal network flexibility enables animals to respond appropriately to changes in their internal and external states. We are using the isolated crab stomatogastric nervous system to determine how extrinsic inputs contribute to network flexibility. The stomatogastric system includes the well-characterized gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the stomatogastric ganglion. Projection neurons with somata in the commissural ganglia (CoGs) regulate these rhythms. Previous work characterized a unique gastric mill rhythm that occurred spontaneously in some preparations, but whose origin remained undetermined. This rhythm includes a distinct protractor phase activity pattern, during which a key gastric mill circuit neuron (LG neuron) and the projection neurons MCN1 and CPN2 fire in a pyloric rhythm-timed activity pattern instead of the tonic firing pattern exhibited by these neurons during previously studied gastric mill rhythms. Here we identify a new extrinsic input, the post-oesophageal commissure (POC) neurons, relatively brief stimulation (30 s) of which triggers a long-lasting (tens of minutes) activation of this novel gastric mill rhythm at least in part via its lasting activation of MCN1 and CPN2. Immunocytochemical and electrophysiological data suggest that the POC neurons excite MCN1 and CPN2 by release of the neuropeptide Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). These data further suggest that the CoG arborization of the POC neurons comprises the previously identified anterior commissural organ (ACO), a CabTRP Ia-containing neurohemal organ. This endocrine organ thus appears to also have paracrine actions, including activation of a novel and lasting gastric mill rhythm.

Antifeeding properties of myosuppressin in a generalist phytophagous leafworm, Spodoptera littoralis (Boisduval)

Insect myosuppressins are a family of peptides with a characteristic HV/SFLRFamide carboxy terminus. They are expressed in brain, neurohemal organs, stomatogastric nervous system, and in midgut endocrine cells. From a functional point of view, myosuppressins inhibit contractions of different visceral muscles, stimulate certain skeletal muscles and activate enzyme secretion from the gut. Moreover, in the omnivorous cockroach Blattella germanica, myosuppressin inhibits food intake. Based on these results, we studied the antifeeding activity of myosuppressin in the phytophagous leafworm Spodoptera littoralis. Firstly, we isolated the cDNA corresponding to the S. littoralis myosuppressin precursor encoding the typical myosuppressin peptide of lepidopterans: pQDVVHSFLRFamide. Then, we determined the expression patterns (in terms of mRNA and peptide) of myosuppressin in brain and midgut, and peptide levels in the haemolymph. Myosuppressin patterns in the brain and haemolymph were similar, and symmetrical to that of food consumption, thus suggesting that myosuppressin might inhibit feeding in S. littoralis. Moreover, synthetic myosuppressin effectively inhibited food intake in non-choice antifeeding tests. Taken together, the obtained results point to the hypothesis that myosuppressin represses feeding in S. littoralis.

Comparative peptidomics of four related hemipteran species: Pyrokinins, myosuppressin, corazonin, adipokinetic hormone, sNPF, and periviscerokinins

We performed the first comprehensive peptidomic analysis of neurohormones from hemipteran insects by analyzing the neuropeptides of two major neurohemal organs, namely the corpora cardiaca and abdominal perisympathetic organs. For the experiments we selected four related species of polyphagous stinkbugs (Pentatomidae), three of which are known to attack several important food crops. Peptide sequences were identified by MALDI-TOF mass spectrometry; tandem fragmentation of myosuppressin, sNPF, CAPA-periviscerokinins and pyrokinins revealed novel sequences not known from other insects so far. Most Leu/Ile and Glu/Lys ambiguities could be solved by either specific side-chain fragmentations or on-plate acetylation experiments. The identification of the specific sequences provides a solid basis for forthcoming pharmacological tests to study the neuroendocrine system of these pest insects. However, it should be mentioned in this context that the sequences of the peptides from different stinkbugs are likely not representative of Hemiptera in general. The forthcoming release of the genome from the reduviid Rhodnius prolixus will provide sufficient data to clear this point.

Windows of the brain: Towards a developmental biology of circumventricular and other neurohemal organs

We review the anatomical and functional features of circumventricular organs in vertebrates and their homologous neurohemal organs in invertebrates. Focusing on cyclostomes (lamprey) and urochordates (ascidians), we discuss the evolutionary origin of these organs as a function of their cell type specification and morphogenesis.

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.

The satiety signaling neuropeptide perisulfakinin inhibits the activity of central neurons promoting general activity

The metabolic state is one of the determinants of the general activity level. Satiety is related to resting or sleep whereas hunger correlates to wakefulness and activity. The counterpart to the mammalian satiety signal cholecystokinin (CCK) in insects are the sulfakinins. The aim of this study was to resolve the mechanism by which the antifeedant activity of perisulfakinin (PSK) in Periplaneta americana is mediated. We identified the sources of PSK which is used both as hormone and as paracrine messenger. PSK is found in the neurohemal organ of the brain and in nerve endings throughout the central nervous system. To correlate the distributions of PSK and its receptor (PSKR), we cloned the gene coding for PSKR and provide evidence for its expression within the nervous system. It occurs only in a few neurons, among them are the dorsal unpaired median (DUM) neurons which release octopamine thereby regulating the general level of activity. Application of PSK to DUM neurons attenuated the spiking frequency (EC50=11pM) due to reduction of a pacemaker Ca2+ current through cAMP-inhibited pTRPγ channels. PSK increased the intracellular cAMP level while decreasing the intracellular Ca2+ concentration in DUM neurons. Thus, the satiety signal conferred by PSK acts antagonistically to the hunger signal, provided by the adipokinetic hormone (AKH): PSK depresses the electrical activity of DUM neurons by inhibiting the pTRPγ channel that is activated by AKH under conditions of food shortage.

Identification of PVK/CAP2b neuropeptides from single neurohemal organs of the stable fly and horn fly via MALDI-TOF/TOF tandem mass spectrometry

MALDI-TOF/TOF tandem mass spectrometry has been applied to determine the complete sequences of the PVK/CAP2b neuropeptides in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans, insect pests of livestock. This peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The sequences are as follows: Stoca-PVK/CAP2b-1, AGGASG L YAFPRVa; Stoca-PVK/CAP2b-2, NAK L YPVPRVa; and Haeir-PVK/CAP2b-1, AGGASG L YAFPRVa; Haeir-PVK/CAP2b-1, NAK L YPMPRVa. Both Stoca-PVK/CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC 50 values between 3 and 11 nM. The identification of these novel neuropeptides adds to our knowledge of the peptidomes of flies, and can aid in the development of neuropeptide-based control strategies of these insect pests.

Mass spectrometric assignment of Leu/Ile in neuropeptides from single neurohemal organ preparations of insects

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF–TOF) tandem mass spectrometry has been applied for the first time on an insect/arthropod target, focusing on PVK/CAP2b neuropeptides in the housefly Musca domestica and flesh fly Neobellieria bullata. The peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The confirmation of side-chain fragments which allows assignment of Leu/Ile even from samples as small as neurohemal organs will greatly accelerate the identification of novel neuropeptides that are implicated in the regulation of critical physiological processes in insects. The unnatural Ile analog is 4.5 times more active than the native Leu sequence in a housefly Malpighian tubule fluid secretion assay, which reinforces the caveat that potency values in a biological assay cannot be relied upon to predict the native sequence.

Identification of a Novel Prothoracicostatic Hormone and Its Receptor in the Silkworm Bombyx mori

The insect brain regulates the activity of the prothoracic glands to secrete ecdysteroids, which affect growth, molting, and metamorphosis. Here we report the identification of a novel prothoracicostatic factor and its receptor in the silkworm Bombyx mori. The prothoracicostatic factor purified from pupal brains of B. mori is a decapeptide with the conserved structure of an insect myosuppressin and thus named Bommo-myosuppressin. Bommo-myosuppressin dose dependently suppressed the cAMP level and inhibited ecdysteroidogenesis in the larval prothoracic glands at much lower concentrations than the prothoracicostatic peptide, the other prothoracicostatic factor reported previously. In vitro analyses using a prothoracic gland incubation method revealed that Bommo-myosuppressin and prothoracicostatic peptide regulate the prothoracic gland activity via different receptors. In situ hybridization and immunohistochemistry revealed the existence of Bommo-myosuppressin in the brain neurosecretory cells projecting to neurohemal organs in which it is stored. We also identified and functionally characterized a specific receptor for Bommo-myosuppressin and showed its high expression in the prothoracic glands. All these results suggest that Bommo-myosuppressin functions as a prothoracicostatic hormone and plays an important role in controlling insect development.

Regulators Acting in Combinatorial Codes Also Act Independently in Single Differentiating Neurons

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, we have resolved how each of these factors contributes to ap neuron diversity. We find that these factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, we also find that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, we show that single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits.