Meet Zandawala

Urbilaterian origin of paralogous GnRH and corazonin neuropeptide signalling pathways

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive maturation in humans and other vertebrates. Homologs of GnRH and its cognate receptor have been identified in invertebrates–for example, the adipokinetic hormone (AKH) and corazonin (CRZ) neuropeptide pathways in arthropods. However, the precise evolutionary relationships and origins of these signalling systems remain unknown. Here we have addressed this issue with the first identification of both GnRH-type and CRZ-type signalling systems in a deuterostome–the echinoderm (starfish) Asterias rubens. We have identified a GnRH-like neuropeptide (pQIHYKNPGWGPG-NH2) that specifically activates an A. rubens GnRH-type receptor and a novel neuropeptide (HNTFTMGGQNRWKAG-NH2) that specifically activates an A. rubens CRZ-type receptor. With the discovery of these ligand-receptor pairs, we demonstrate that the vertebrate/deuterostomian GnRH-type and the protostomian AKH systems are orthologous and the origin of a paralogous CRZ-type signalling system can be traced to the common ancestor of the Bilateria (Urbilateria).

DINeR: Database for Insect Neuropeptide Research

Neuropeptides are responsible for regulating a variety of functions, including development, metabolism, water and ion homeostasis, and as neuromodulators in circuits of the central nervous system. Numerous neuropeptides have been identified and characterized. However, both discovery and functional characterization of neuropeptides across the massive Class Insecta has been sporadic. To leverage advances in post-genomic technologies for this rapidly growing field, insect neuroendocrinology requires a consolidated, comprehensive and standardised resource for managing neuropeptide information. The Database for Insect Neuropeptide Research (DINeR) is a web-based database-application used for search and retrieval of neuropeptide information of various insect species detailing their isoform sequences, physiological functionality and images of their receptor-binding sites, in an intuitive, accessible and user-friendly format. The curated data includes representatives of 50 well described neuropeptide families from over 400 different insect species. Approximately 4700 FASTA formatted, neuropeptide isoform amino acid sequences and over 200 records of physiological functionality have been recorded based on published literature. Also available are images of neuropeptide receptor locations. In addition, the data include comprehensive summaries for each neuropeptide family, including their function, location, known functionality, as well as cladograms, sequence alignments and logos covering most insect orders. Moreover, we have adopted a standardised nomenclature to address inconsistent classification of neuropeptides. As part of the H2020 nEUROSTRESSPEP project, the data will be actively maintained and curated, ensuring a comprehensive and standardised resource for the scientific community. DINeR is publicly available at the project website: http://www.neurostresspep.eu/diner/.

Systemic corazonin signalling modulates stress responses and metabolism in Drosophila

Stress triggers cellular and systemic reactions in organisms to restore homeostasis. For instance, metabolic stress, experienced during starvation, elicits a hormonal response that reallocates resources to enable food search and readjustment of physiology. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. Here we show that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila. The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the flys resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, our findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. Our study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales.

Localization of Neuropeptide Gene Expression in Larvae of an Echinoderm, the Starfish Asterias rubens

Neuropeptides are an ancient class of neuronal signaling molecules that regulate a variety of physiological and behavioral processes in animals. The life cycle of many animals includes a larval stage(s) that precedes metamorphic transition to a reproductively active adult stage but, with the exception of Drosophila melanogaster and other insects, research on neuropeptide signaling has hitherto largely focused on adult animals. However, recent advances in genome/transcriptome sequencing have facilitated investigation of neuropeptide expression/function in the larvae of protostomian (e.g., the annelid Platynereis dumerilii) and deuterostomian (e.g., the urochordate Ciona intestinalis) invertebrates. Accordingly, here we report the first multi-gene investigation of larval neuropeptide precursor expression in a species belonging to the phylum Echinodermata—the starfish Asterias rubens. Whole-mount mRNA in situ hybridization was used to visualize in bipinnaria and brachiolaria stage larvae the expression of eight neuropeptide precursors: L-type SALMFamide (S1), F-type SALMFamide (S2), vasopressin/oxytocin-type, NGFFYamide, thyrotropin-releasing hormone-type, gonadotropin-releasing hormone-type, calcitonin-type and corticotropin-releasing hormone-type. Expression of only three of the precursors (S1, S2, NGFFYamide) was observed in bipinnaria larvae but by the brachiolaria stage expression of all eight precursors was detected. An evolutionarily conserved feature of larval nervous systems is the apical organ and in starfish larvae this comprises the bilaterally symmetrical lateral ganglia, but only the S1 and S2 precursors were found to be expressed in these ganglia. A prominent feature of brachiolaria larvae is the attachment complex, comprising the brachia and adhesive disk, which mediates larval attachment to a substratum prior to metamorphosis. Interestingly, all of the neuropeptide precursors examined here are expressed in the attachment complex, with distinctive patterns of expression suggesting potential roles for neuropeptides in the attachment process. Lastly, expression of several neuropeptide precursors is associated with ciliary bands, suggesting potential roles for the neuropeptides derived from these precursors in control of larval locomotion and/or feeding. In conclusion, our findings provide novel perspectives on the evolution and development of neuropeptide signaling systems and neuroanatomical insights into neuropeptide function in echinoderm larvae.

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. Here, we investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. We also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. Our data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.

A single pair of leucokinin neurons are modulated by feeding state and regulate sleep-metabolism interactions

Dysregulation of sleep and feeding has widespread health consequences. Despite extensive epidemiological evidence for interactions between sleep and metabolic function, little is known about the neural or molecular basis underlying the integration of these processes. Drosophila melanogaster potently suppress sleep in response to starvation, and powerful genetic tools allow for mechanistic investigation of sleep-metabolism interactions. We have previously identified neurons expressing the neuropeptide leucokinin (Lk) as being required for starvation-mediated changes in sleep. Here, we demonstrate an essential role for Lk neuropeptide in metabolic regulation of sleep. Further, we find that the activity of Lk neurons is modulated by feeding state and circulating nutrients, with reduced activity in response to glucose and increased activity under starvation conditions. Both genetic silencing and laser-mediated microablation localize Lk-mediated sleep regulation to a single pair of Lk neurons within the lateral horn (LHLK) that project near primary sleep and metabolic centers of the brain. A targeted screen identified a critical role for AMP-activated protein kinase (AMPK) in starvation-modulated changes in sleep. Disruption of AMPK function in Lk neurons suppresses sleep and increases LHLK activity in fed flies, phenocopying the starvation state. Taken together, these findings localize feeding state-dependent regulation of sleep to a single pair of neurons within the fruit fly brain and provide a system for investigating the cellular basis of sleep-metabolism interactions.

Orchestration of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin

Behavior and physiology are orchestrated by neuropeptides acting as neuromodulators and/or circulating hormones. A central question is how these neuropeptides function to coordinate complex and competing behaviors. The neuropeptide leucokinin (LK) modulates diverse functions, including circadian rhythms, feeding, water homeostasis, and sleep, but the mechanisms underlying these complex interactions remain poorly understood. Here, we delineate the LK circuitry that governs homeostatic functions that are critical for survival. We found that impaired LK signaling affects diverse but coordinated processes, including regulation of stress, water homeostasis, locomotor activity, and metabolic rate. There are three different sets of LK neurons, which contribute to different aspects of this physiology. We show that the calcium activity of abdominal ganglia LK neurons (ABLKs) increases specifically following water consumption, but not under other conditions, suggesting that these neurons regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of the LK receptor (Lkr), mined brain single-cell transcriptome dataset for genes coexpressed with Lkr, and utilized trans-synaptic labeling to identify synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and sensory cells, and the post-synaptic signal in sensory neurons, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) in IPCs and modulated stress responses. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons orchestrate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating neuroendocrine regulation of behavior and brain-to-periphery communication.

Ophiuroid Phylotranscriptomics Enables Discovery Of Novel Echinoderm Representatives Of Bilaterian Neuropeptide Families And Reconstruction Of Neuropeptide Precursor Evolution Over ~270 Million Years

Neuropeptides are a diverse class of intercellular signaling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signaling. Here detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over thirty bilaterian neuropeptide precursor families were identified, some of which occur as paralogs. Furthermore, homologs of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally-related, neuropeptides. Here we performed an unprecedented investigation of the evolution of neuropeptide copy-number over a period of ~270 million years by analysing sequence data from over fifty ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide “cocktails” is functionally important, but with plasticity over long evolutionary time scales.Background: Neuropeptides are a diverse class of intercellular signaling molecules that mediate neuronal regulation of many physiological and behavioural processes, including feeding, reproduction and locomotion. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signaling. Here we report a phylo-transcriptomic analysis of neuropeptide precursors in over fifty species of brittle stars (Class Ophiuroidea; Phylum Echinodermata). Results: Detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, enabled the first comprehensive identification of neuropeptide precursors in ophiuroids. Representatives of over thirty bilaterian neuropeptide precursor families were identified, some of which occur as paralogs (e.g. thyrotropin-releasing hormone, corticotropin-releasing hormone, cholecystokinin, somatostatin and pedal peptide). Furthermore, homologs of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally-related, neuropeptides. Here we performed an unprecedented investigation of the evolution of neuropeptide copy-number over a period of ~270 million years by analysing sequence data from over fifty ophiuroid species, with reference to a robust phylogeny. Interestingly, the number of neuropeptide copies in the majority of precursors was constant across all the species examined, but examples of clade-specific losses/gains of neuropeptides were also observed. Conclusions: We report here the most comprehensive analysis to date of neuropeptide precursors in the phylum Echinodermata, with novel representatives of several bilaterian neuropeptide families discovered for the first time in echinoderms. Furthermore, analysis of precursor proteins comprising multiple copies of identical or related neuropeptides across ~270 million years of ophiuroid evolution indicates that the composition of neuropeptide cocktails is functionally important, but with plasticity over long evolutionary time scales.