MELANOPSIN SIGNALING IN MOUSE IRIS by Qian Wang A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland November, 2016 © 2016 Qian Wang All Rights Reserved Abstract The pupil size is determined by an antagonistic action between the circumferential sphincter muscle and the radial dilator muscle in the iris. Upon illumination, the pupils in both eyes constrict to a broadly similar extent because of the bilateral reflex pathways through the brain. Despite this widely-accepted picture, it has been reported that, the isolated iris sphincter muscle of sub- primate nocturnal and crepuscular mammals generally shows, surprisingly, a light-induced contraction. In mouse, this local iridic light effect requires the pigment, melanopsin, the localization and signaling mechanism of which remains unclear. Others have suggested that the local light reflex of the isolated mouse iris sphincter results merely from collateral axons of intrinsically-photosensitive retinal ganglion cells (ipRGCs) forming presynaptic terminals on parasympathetic muscarinic innervation of the sphincter muscle cells. Here, we exclude this model by showing that atropine, a muscarinic blocker, eliminated the effect of acetylcholine (ACh) on the isolated mouse sphincter muscle without affecting its light response, indicating that the light-induced contraction of the isolated iris sphincter is independent of muscarinic synaptic transmission. By genetic labeling, we underpinned melanopsin’s expression in about 10% of mouse iris-sphincter muscle cells, in which light and acetylcholine appear to signal through shared, albeit not identical pathways. The response of iris sphincter muscle isolated from the conditional Gαq Gα11 double-knockout line to ACh was practically the same as control, but the muscle’s response to a light flash was significantly reduced. ii Knockout of genes encoding other members (i.e. Gα14 or Gα15) from Gq/11- subfamily did not affect the muscle’s response to ACh or light, indicating that melanopsin signals to both Gαq and Gα11 in the iris muscle cells. Iris sphincter isolated from Plcβ2-/- animal also showed defective response to light flashes, but the response to ACh stimulation was largely preserved, suggesting that different sets of PLCβs are expressed in light-responsive versus light-irresponsive sphincter muscle cells. Knockout of Itpr1 eliminated both light- and ACh-induced muscle contraction, indicating that the response to both stimuli relies on release of Ca2+ from the intracellular store. The overall conclusion from this work is that the light induced muscle contraction is generated from a subpopulation of sphincter muscle cells that express melanopsin. The contractions of the iris sphincter muscle elicited by ACh and by light are via separate signaling pathways, although these pathways converge at the step of actin-myosin interaction triggered by a rise in intracellular Ca2+ concentration. This study generated the first molecular and genetic analysis of the iris-based photoreceptors, expanded our knowledge on the mechanism of photoreception mediated by melanopsin. iii Acknowledgements I would like to express my deepest gratitude to my advisor Dr. King-Wai Yau, for his continuous support and guidance during my study at Hopkins. I also wish to thank my committee member, Dr. Xinzhong Dong, Dr. Jeremy Nathans, and Dr. Leslie Tung, for their tremendous help during the past five years of my graduate studies. I would like to extend my gratitude to my current and former colleagues, Dr. Tian Xue, Dr. Donggen Luo, Dr. Lihui Cao, Dr. Zheng Jiang, Dr. Xiaozhi Ren, Dr. Rongchang Li, Dr. Chih-Chun Lin, Dr. Wendy Yue, Dr. Xiaojun Li, Yanghui Sheng, Daniel Silverman, Lujing Chen, and Liusong Ding, for their great support and helpful suggestions on my research project. Finally, I would like to thank my parents, mother Xiaofan Zhao, father Jinxing Wang, for their love and support. I wish to share my happiness and proud of being graduating with them. iv Table of Contents 1. Introduction .................................................................................................... 1 1.1 General structure of the mammalian iris .................................................. 1 1.2 Musculature: sphincter pupillae and dilator pupillae ................................ 1 1.3 Innervation and control of the mammalian iris smooth muscle ................ 2 1.4 Receptors in iris sphincter and dilator pupillae and pupillary size regulation .......................................................................................................... 4 1.4.1 Muscarinic receptors in sphincter and dilator pupillae....................... 4 1.4.2 Intracellular signaling by muscarinic receptors ................................. 6 1.4.3 Adrenergic receptors in sphincter and dilator pupillae ...................... 7 1.5 Photosensitivity of the iris muscle cells.................................................... 8 1.5.1 Discovery of the photomechanical response (PMR) ......................... 9 1.5.2 PMR in mammals ............................................................................ 11 1.5.3 The relationship between iPLR and neural control of PLR .............. 12 1.5.4 Light responsive protein in mammalian iris ..................................... 13 1.5.5 Cellular and Molecular mechanism of the iPLR in mouse ............... 14 1.5.6 PMR in birds ................................................................................... 15 1.6 Objective ............................................................................................... 17 v 2. Experimental Procedures ............................................................................. 18 2.1 Animals .................................................................................................. 18 2.2 Generation of the Opn4-Cre line (By Wendy Yue, Laboratory of King-Wai Yau) 18 2.3 Generation of the floxed-opn4 line ........................................................ 20 2.4 Solutions ................................................................................................ 20 2.5 Iris sphincter muscle force measurement .............................................. 21 2.6 In situ pupillometry ................................................................................ 23 2.7 Immunohistochemistry ........................................................................... 24 2.8 Quantitative RT-PCR ............................................................................. 25 2.9 Knockdown of chicken cryptochrome 1 and 2 (cCry1 and 2) by morpholino-modified oligonucleotides ............................................................. 26 2.10 Cloning and expression of cCry1 and cCry2 ...................................... 27 3. Melanopsin signaling in mouse iris .............................................................. 31 3.1 ACh and light-induced contraction of isolated mouse iris sphincter muscle ............................................................................................................. 31 3.2 Light-Induced contraction of isolated sphincter muscle is independent of cholinergic transmission .................................................................................. 33 3.3 Melanopsin-expression in mouse iris .................................................... 35 3.4 Tissue-specific ablation of melanopsin-expression in smooth muscle ... 37 vi 3.5 Melanopsin signaling mechanism in sphincter muscle .......................... 39 3.6 Discussion ............................................................................................. 41 4. iPLR in Chicken ........................................................................................... 76 4.1 The photoreceptive role of cryptochrome in chicken iris ........................ 76 4.2 Summary and future direction ............................................................... 78 Attachment-Sequence of floxed-Opn4 allele ...................................................... 85 References ......................................................................................................... 97 CURRICULUM VITAE FOR Ph.D. CANDIDATES…………………………...…..109 vii List of Figures Figure 2-1 Force sensor and calibration…………………………………………….30 Figure 3-1 Segregation of ACh- and light-signaling pathways in iris sphincter muscle ................................................................................................................ 47 Figure 3-2 Involvement of gap junctional coupling in light-induced contraction of isolated iris sphincter .......................................................................................... 49 Figure 3-3 Light responses of isolated iris sphincter muscle with or without ACh exposure ............................................................................................................. 51 Figure 3-4 Effect of TTX and atropine on iPLR ................................................... 53 Figure 3-5 Effect of Glutamate and PACAP receptor blocker on iPLR ............... 55 Figure 3-6 Expression of Opn4S and Opn4L in the mouse iris and retina .......... 57 Figure 3-7 X-gal staining of iris and retina isolated from the Opn4τlacZ/+ line ....... 59 Figure 3-8 Specificity of the Opn4-Cre line in mouse retina ............................... 60 Figure 3-9 Melanopsin expression in mouse iris................................................. 62 Figure 3-10 Melanopsin-expression in the mouse