The Journal fetal light response regulates mouse eye development - PDF document

3/12/2013 A direct and melanopsin-dependent The Journal fetal light response regulates mouse eye development Sujata Rao, Christina Chun, Jieqing Fan, J Matthew Kofron, Michael B Yang, Rashmi S Hegde,Napoleone Ferrara, David R Copenhagen, Richard A Lang S Nature: “is the world's most highly Nature (2013), PMID 23334418 cited interdisciplinary science journal” NEUROSCIENCE JOURNAL CLUB BIONB 4110 S Published weekly CORNELL UNIVERSITY S Impact Factor 36.101 Glen Malaret Nishad Prinja S Nature Publishing Group S Source: http://www.nature.com/nature/journal/v494/n7436/nature11823/metrics The Institutions The People S Sujata Rao, PhD S Cornell Alumna in NBB S Research Associate at Center of health sciences One of the top pediatric S S Cincinnati Children’s Lang research, patient care and hospitals in the nation Laboraty education S Receives second most NIH One of ten campuses in the UC funds for a children’s hospital S S Jieqing Fan S Christina Chun System Cincinnati Children’s Research S Research Associate at UCSF S PhD Candidate S S Only one exclusively dedicated Foundation Copenhagen lab University of Cincinnati S to the health sciences School of Medicine 1

3/12/2013 S J. Matthew Kofrom, PhD PhD University of Minnesota S S Rashmi S. Hegde, PhD S Napoleone Ferrara, Assistant Professor, UC S MD PhD University of S S Michael B. Yang, MD Department of Pediatrics Pittsburgh S Genetech Fellow MD Harvard Medical S S Professor, UC Department S Professor at UC San School of Pediatrics Diego School of Medicine S Associate Professor, UC Department of S Credited with identifying Ophthalmology the human VEGF gene S David R. Copenhagen PhD S Richard A. Lang, PhD PhD UC Berkley S PhD University of S Professor of S Melbourne Ophthalmology and Director of the Visual S Physiology at UCSF Systems Group in S Research Supervisor Cincinnati Children’s Professor, UC Department S of Pediatrics Research Supervisor S Special thanks to Dr. Howland for info & images 2

3/12/2013 Human hyaloid vasculature usually regresses at about 7 th month of pregnancy. Here, in a premature human infant the tunical vasculosa lentis surrounds the lens (arrows 1) and is contiguous with the hyaloid vascular system (arrow 2). Notice the glial sheath of the hyaloid Vitreoretinal findings similar to retinopathy of prematurity in infants with compound heterozygous protein S deficiency ☆ Mintz-Hittner, et al. (1999) Ophthalmology Volume 106, Issue 8, 1 August 1999, Pages 1525 – 1530 artery (arrow 3). From: www.missionforvisionusa.org Hypothesis|Scope Figure S7 S Hypothesis A pathway exists – light responsiveness in early retina triggers the clearance of optic axis. S Scope S Investigating – light/dark response, gene expression of Opn4 , mRNA coding of VEGFA, mother-pup development template. Methods: Cell Sorting Methods: Hyaloid/Retinal Labelling Flow Sorting w/ retinal neuron S markers S Hyaloid Vessels collected and Hoechst stained (Blue Dye) S Cells labeled with Goat PDGFR S Flat-mounts were prepared and labeled with antibodies S Cells sorted with FACSAriaII S Vessels were counted in many x200 microscope fields 3

3/12/2013 Figure 2: Hyaloid regression and retinal angiogenesis are Figure 1: Hyaloid regression is regulated by light. regulated by melanopsin. Method: Figure S5 Isolation & Analysis of Vitreous S Eyes washed - Phosphate Buffered Saline (PBS) rinse S Neutral, sterile buffer free of ionic contents S Small slit thru retina – vitreous collected S ELISA performed – Vegfa Quantikine kits S Immunoblots probed – unique VEGFA carboxy-terminal antibody Method: Figure 3; a-e RNA isolation & qPCR S RNA isolated – RNeasy S qPCR performed w. QuantiTect SYBR green S Both agents supplied by Qiagen Labs Inc. S Actin amplified for normalization S qPCR performed w. Vegfa primers – > S 5’ – GACAGAACAAAGCCAGA – 3’ S 5’ – CACCGCCTTGGCTTGTCAC – 3’ 4

3/12/2013 Method: Figure 3; f-i Assessment of Hypoxia S Pups inj. w. 180µg pimonidazole HCl (hypoxyprobe) ea. S Hypoxyprobe + rabbit 1º antibody + anti-rabbit 2º Alexa594 S (fluorescent labeling agents) S Pixel intensity S Averaged along line – from retinal center to periphery S 20-25 line intervals per retina, 5-6 retinas per genotype Methods: Light Measurments Figure S6 S Controlled for light intensity S Converting wavelengths into units of photons cm -2 s -1 S λ =479nm (Blue light) calculated to be 5 𝑦 10 13 photons cm -2 s -1 S Suns radiant flux density: 2.6 𝑦 10 16 photons cm -2 s -1 S LED lights were inserted into the mice’s abdominal cavity Figure 4 Questions? 5

3/12/2013 Thank you to Dr. Hopkins, Dr. Howland & the members of NSJ Cornell Fin 6