The Shaughnessy Lab takes a broad approach to studying osmoregulatory and stress physiology and the endocrine programs which control them. We integrate investigations at the molecular, cellular, organ, and organismal levels to gain mechanistic insights into physiological function. Our research can be applied in evolutionary, ecological, and translational contexts. Please learn more about the various research tracts in the Shaughnessy Lab below.
Activation Pathways of CFTR-Mediated Ion Transport in the Human Airway
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations of the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator) that render the mature CFTR protein dysfunctional. Expressed in the ‘pulmonary ionocyte’ as well as other cell types, CFTR is an apical, cAMP- and phosphorylation-activated Cl- channel that is critical for maintaining osmoregulatory homeostasis of airway and intestinal epithelia, allowing for proper mucociliary clearance and organ function. Recent progress in the treatment of CF rests on the discovery of small-molecule drugs (CFTR ‘modulators’) to aid in folding and trafficking (‘correctors’, ‘enhancers’, and ‘amplifiers’) and channel gating and conductance (‘potentiators’) of mutant CFTR. These CFTR modulators are remarkably effective when used in combination, yet still only partially restore mutant CFTR function, and treatment with modulators still relies on endogenous levels of intracellular cAMP to regulate the activity of the modulator-rescued CFTR.
In addition to basic studies on the ion channel properties of CFTR, the Shaughnessy Lab conducts pre-clinical research exploring the incorporation of a new class of CFTR modulators, CFTR ‘activators’, into the regular combination drug treatment regimens already being used to treat CF. Specifically, we use in vitro models of the human airway epithelium to investigate clinically feasible mechanisms of CFTR activation via agonizing G protein-coupled receptors that stimulate intracellular cAMP levels in airway epithelial cells.
Techniques used in this research track could include mammalian cell culture, air-liquid interface cell culture, transfection, RNA interference, in vitro pharmacology, gene expression analyses (PCR, real-time PCR, in situ hybridization), immunological analyses (Western blotting, co-immunoprecipitation, immunofluorescence microscopy), enzyme-linked immunosorbent assay (ELISA), transactivation (luciferase reporter gene) assays, and electrophysiology (Ussing chamber).
Functional Evolution of Ion Transporters and Ionoregulatory Physiology
Salt-secreting epithelia in vertebrate animals exist in a variety of organs, including the elasmobranch rectal gland, actinopterygian gill, amphibian skin, and reptilian lingual and avian nasal salt glands. These organs, although disparate and non-homologous, leverage a remarkably similar cell type to achieve Cl- secretion. The defining feature of the salt-secreting ionocyte in these tissues is the co-expression of two basolateral ion transporters, Na+/K+-ATPase (Nka) and Na+/K+/2Cl- cotransporter (Nkcc1), along with an apical Cl- channel, the cystic fibrosis transmembrane conductance regulator (Cftr). In these ionocytes, Cftr is the primary conduit for Cl- secretion.
This Nka-, Nkcc1-, and Cftr-rich ionocyte was first characterized in the shark rectal gland and later in the marine actinopterygian fish gill as the key cellular pathway for Cl- secretion that is essential to the maintenance of ionic homeostasis and survival in seawater (SW). Since its first description in elasmobranchs, all ionoregulating marine fishes that have been examined to date exhibit a Cftr-rich ionocyte as the critical mechanism responsible for Cl- secretion. Thus, it has been presumed that all marine fishes require a Cftr-rich Cl-secretory ionocyte to survive in the marine environment. More recently, single-cell RNA sequencing of the human and murine airways led to the discovery of a strikingly similar pathway for Cl- secretion via an Nka-, Nkcc1-, and Cftr-rich cell type, the ‘pulmonary ionocyte’.
The Shaughnessy Lab takes an integrative approach at understanding the molecular, cellular, epithelial, physiological, and endocrine mechanisms animals use to osmo- and ionoregulate and how these mechanisms evolved. We focus this effort primarily on understanding how basal marine fishes (including hagfishes, lampreys, elasmobranchs, and sturgeon) osmo- and ionoregulate in the highly salt-concentrated environment. We combine molecular structure/functions studies of ion transporters in vitro with physiological investigations in vivo using live animals models.
Experimental approaches used in this research track could include trips to freshwater/marine stations, live animal experimentation, mammalian cell culture, non-mammalian cell culture, transfection, RNA interference, in vitro, ex vivo, and in vivo pharmacology, gene expression analyses (PCR, real-time PCR, in situ hybridization), immunological analyses (Western blotting, immunofluorescence microscopy), enzyme-linked immunosorbent assay (ELISA), radioactive isotope assays (radio-immunoassays, radio-receptor binding assays), transactivation (luciferase reporter gene) assays, electrophysiology (Ussing chamber), and bioinformatics (RNAseq, scRNAseq, computational evolutionary biology).
The Ancient Origins and Early Evolution of Vertebrate 'Stress Physiology'
The hypothalamic-pituitary-adrenal/interrenal (HPA/HPI) axis is a highly conserved endocrine axis among vertebrates that regulates corticosteroid biosynthesis in adrenal/interrenal tissue in response to stress. Key to this pathway is the production of adrenocorticotropic hormone (ACTH) from a precursor protein pro-opiomelanocortin (POMC). Upon release from the pituitary into circulation, ACTH binds the melanocortin 2 receptor (Mc2r), which is almost exclusively expressed in corticosteroidogenic tissue such as the adrenal/interrenal. Mc2r is a G-protein coupled receptor and
its activation by ACTH causes a cAMP-dependent, intracellular signaling cascade that results in the transcriptional upregulation and phosphorylation of steroidogenic acute regulatory protein (star) and the initiation of corticosteroid biosynthesis. This complex endocrine circuit exists in near identical arrangement in every vertebrate lineage except the most basal vertebrate group, Agnatha (including hagfishes and lampreys).
The Shaughnessy Lab conducts in vitro, ex vivo, and in vivo studies to investigate corticosteroidogenic endocrine pathways and the relationships between adrenocorticotropic pituitary hormones and their receptors in these ancient vertebrate lineages (hagfishes and lampreys) and protochordates (e.g., amphioxus). Additionally, we use phylogenetic modeling to reconstruct and examine the hypothetical ancestral hormone-receptor relationships. In doing so, we mechanistically explore how this complex endocrine circuit arose on an evolutionary timescale, particularly at the dawn of the chordate phylum.
Experimental approaches used in this research track could include trips to freshwater/marine stations, live animal experimentation, mammalian cell culture, non-mammalian cell culture, transfection, RNA interference, in vitro, ex vivo, and in vivo pharmacology, gene expression analyses (PCR, real-time PCR, in situ hybridization), immunological analyses (Western blotting, immunofluorescence microscopy), enzyme-linked immunosorbent assay (ELISA), radioactive isotope assays (radio-immunoassays, radio-receptor binding assays), and transactivation (luciferase reporter gene) assays, and bioinformatics (RNAseq, computational phylogenetics).