Transgenic Drosophila lines for LexA-dependent gene and growth regulation.
G3 (Bethesda, Md.)
Conditional expression of short hairpin RNAs (shRNAs) with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of two independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-shRNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based shRNA expression vector to achieve shRNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.
View details for DOI 10.1093/g3journal/jkac018
View details for PubMedID 35100369
- Why earlier may be better: a look at the use of metabolic and bariatric surgery in the treatment of severe childhood obesity. Surgery for obesity and related diseases : official journal of the American Society for Bariatric Surgery 2021
Discovering signaling mechanisms governing metabolism and metabolic diseases with Drosophila.
There has been rapid growth in the use of Drosophila and other invertebrate systems to dissect mechanisms governing metabolism. New assays and approaches to physiology have aligned with superlative genetic tools in fruit flies to provide a powerful platform for posing new questions, or dissecting classical problems in metabolism and disease genetics. In multiple examples, these discoveries exploit experimental advantages as-yet unavailable in mammalian systems. Here, we illustrate how fly studies have addressed long-standing questions in three broad areas-inter-organ signaling through hormonal or neural mechanisms governing metabolism, intestinal interoception and feeding, and the cellular and signaling basis of sexually dimorphic metabolism and physiology-and how these findings relate to human (patho)physiology. The imaginative application of integrative physiology and related approaches in flies to questions in metabolism is expanding, and will be an engine of discovery, revealing paradigmatic features of metabolism underlying human diseases and physiological equipoise in health.
View details for DOI 10.1016/j.cmet.2021.05.018
View details for PubMedID 34139200
Pediatric Metabolic and Bariatric Surgery.
The Surgical clinics of North America
2021; 101 (2): 199–212
Childhood obesity can lead to comorbidities that cause significant decrease in health-related quality of life and early mortality. Recognition of obesity as a disease of polygenic etiology can help deter implicit bias. Current guidelines for treating severe obesity in children recommend referral to a multidisciplinary treatment center that offers metabolic and bariatric surgery at any age when a child develops a body mass index that is greater than 120% of the 95th percentile. Obesity medications and lifestyle counseling about diet and exercise are not adequate treatment for severe childhood obesity. Early referral can significantly improve quality and quantity of life.
View details for DOI 10.1016/j.suc.2020.12.007
View details for PubMedID 33743964
Preoperative considerations for the pediatric patient undergoing metabolic and bariatric surgery.
Seminars in pediatric surgery
2020; 29 (1): 150890
To ensure successful outcomes in pediatric patients with severe obesity who undergo metabolic and bariatric surgery (MBS), a number of pre-operative patient management options should be considered. This manuscript will review the indications and contraindications of MBS and special considerations for youth who might benefit from MBS. The treatment team conducts a thorough pre-operative evaluation, assessing risks and benefits of surgical intervention, and prepares patients and families to be successful with MBS by providing education about the surgical intervention and lifestyle changes that will be necessary. This article reviews the pre-operative considerations for adolescents with severe obesity who are being considered for MBS, based upon recent clinical practice guidelines.
View details for DOI 10.1016/j.sempedsurg.2020.150890
View details for PubMedID 32238283
Recruitment of Spinoparabrachial Neurons by Dorsal Horn Calretinin Neurons
2019; 28 (6): 1429-+
The dorsal horn of the spinal cord is the first integration site of somatosensory inputs from the periphery. In the superficial layers of the dorsal horn, nociceptive inputs are processed by a complex network of excitatory and inhibitory interneurons whose function and connectivity remain poorly understood. We examined the role of calretinin-expressing interneurons (CR neurons) in such processing and show that they receive direct inputs from nociceptive fibers and polysynaptic inputs from touch-sensitive Aβ fibers. Their activation by chemogenetic or optogenetic stimulation produces mechanical allodynia and nocifensive responses. Furthermore, they monosynaptically engage spinoparabrachial (SPb) neurons in lamina I, suggesting CR neurons modulate one of the major ascending pain pathways of the dorsal horn. In conclusion, we propose a neuronal pathway in which CR neurons are positioned at the junction between nociceptive and innocuous circuits and directly control SPb neurons in lamina I.
View details for DOI 10.1016/j.celrep.2019.07.048
View details for Web of Science ID 000478978200006
View details for PubMedID 31390558
Connexin36 localization to pinealocytes in the pineal gland of mouse and rat
EUROPEAN JOURNAL OF NEUROSCIENCE
2017; 45 (12): 1594–1605
Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36-containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase-positive and 5-hydroxytryptamine-positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens-1 (ZO-1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36-containing gap junctions that also incorporate the scaffolding protein ZO-1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36-containing gap junctions, which may now be tested by the use of Cx36 null mice.
View details for DOI 10.1111/ejn.13602
View details for Web of Science ID 000403712000009
View details for PubMedID 28474748
View details for PubMedCentralID PMC5507615
Immunofluorescence reveals unusual patterns of labelling for connexin43 localized to calbindin-D28K-positive interstitial cells in the pineal gland
EUROPEAN JOURNAL OF NEUROSCIENCE
2017; 45 (12): 1553–69
Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 μm) or as unusually large pools of Cx43 ranging up to 4-7 μm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes.
View details for DOI 10.1111/ejn.13578
View details for Web of Science ID 000403712000006
View details for PubMedID 28394432