In vivo characterization of Ca2+ dynamics in pancreatic β-cells of zebrafish
PhD student: Luis Delgadillo
Supervisor at TUD: Nikolay Ninov
Supervisor at KCL: Timothy Pullen
Start date: 01.02.2016 Date of defense: 17.11.2020 Dr.rer.medic.
Glucose homeostasis is fundamental for all living organisms. In vertebrates, the hormone insulin regulates the metabolism of carbohydrates, fats and proteins. In order to sustain the glucose homeostasis, the pancreatic β-cells, which produce and secrete insulin, must coordinate their efforts to secrete the right amounts of insulin required by the organism. In vitro studies, have suggested that a subpopulation of β-cells, referred to as “hub-cells”, coordinate islet Ca2+ dynamics during insulin secretion. However, it is unclear whether the hub-cell model pertains to an in vivo scenario, where the islet is densely vascularized and innervated. In this thesis, we employed the genetically-encoded calcium indicator GCaMP6, confocal imaging and optogenetics, to characterize the Ca2+ dynamics of the zebrafish β-cells in vivo.
We found that pancreatic β-cells present endogenous Ca2+ spikes in vivo under basal conditions. These Ca2+ spikes are rapidly suppressed after lowering glucose levels via insulin administration. In addition, the temporal inhibition of blood flow decreases the Ca2+ spikes, suggesting that β-cells are systemically connected. Furthermore, β-cells show a synchronized response to a pericardial glucose injection. Specifically, we found that Ca2+ spikes originate and emanate from a subset of β-cells that are the first to respond to a glucose stimulus. We define these cells as “leader-cells”. We tested if these cells could coordinate the islet in vivo by employing 2-photon laser ablation. Whereas ablation of control cells had no significant effect on the amplitude and duration of the subsequent Ca2+ spikes responses, ablation of leader cells led to a reduction in the Ca2+ response. Furthermore, we developed systems for optogenetic interrogation of β-cells in vivo. We show that the light-gated Cl- ion pump halorhodopsin (NpHR) can be applied to inhibit β-cell depolarization in the zebrafish. We also present the optically orthogonal system of the red Ca2+ indicator K-GECO1 in combination with the blue-shifted channelrhodopsin CheRiff to activate individual β-cell in vivo. Using these new tools, we provide examples where the activation of individual β-cells showed heterogeneous potential to trigger influx of Ca2+ in the rest of the β-cells.
Overall, our results led us to propose a hierarchical model of islet coordination. In contrast to the majority of β-cells, which occupy the bottom of the hierarchy since they present low capability to recruit other cells, the leader cells occupy the top levels, being capable to coordinate a majority of the islets β-cells.
Publications:
Adrenal cortical and chromaffin stem cells: Is there a common progeny related to stress adaptation? C. Steenblock, M.F. Rubin de Celis, A. Androutsellis-Theotokis, M. Sue, L.F. Delgadillo Silva, G. Eisenhofer, C.L. Andoniadou, S.R. Bornstein. Mol Cell Endocrinol. 2017;441:156-163.
GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer. A. Checa-Rojas, L.F. Delgadillo-Silva, M.D.C. Velasco-Herrera, A. Andrade-Dominguez, J. Gil, O. Santillan, L. Lozano, A. Toledo-Leyva, A. Ramirez-Torres, P. Talamas-Rohana, S. Encarnacion-Guevara. Oncotarget. 2018;9:21696-21714.
Isolation and characterization of adrenocortical progenitors involved in the adaptation to stress. C. Steenblock, M.F. Rubin de Celis, L.F. Delgadillo Silva, V. Pawolski, A. Brennand, M. Werdermann, I. Berger, A. Santambrogio, M. Peitzsch, C.L. Andoniadou, A.V. Schally, S.R. Bornstein. Proc Natl Acad Sci U S A. 2018;115:12997-13002.
Modelling pancreatic beta-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection. L.F. Delgadillo-Silva, A. Tsakmaki, N. Akhtar, Z.J. Franklin, J. Konantz, G.A. Bewick, N. Ninov. Dis Model Mech. 2019;12:dmm036004.
Leader beta-cells coordinate Ca(2+) dynamics across pancreatic islets in vivo. V. Salem*, L.D. Silva*, K. Suba, E. Georgiadou, S. Neda Mousavy Gharavy, N. Akhtar, A. Martin-Alonso, D.C.A. Gaboriau, S.M. Rothery, T. Stylianides, G. Carrat, T.J. Pullen, S.P. Singh, D.J. Hodson, I. Leclerc, A.M.J. Shapiro, P. Marchetti, L.J.B. Briant, W. Distaso, N. Ninov, G.A. Rutter. Nat Metab. 2019;1:615-629.
Insights on β-cell regeneration from the zebrafish shoal: from generation of cells to functional integration. P. Chawla, L.F.D. Silva, N. Ninov. Curr Opin Physiol. 2020;14:27-34.
c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair. S.S. Deville, L.F. Delgadillo Silva, A. Vehlow, N. Cordes. Int J Mol Sci. 2020;21:7277.
Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate in vivo β-Cell Function. L. F. Delgadillo-Silva, M. N. Akhtar, E. Taşöz, N. Ninov. J Vis Exp. 2021.