Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Kevin  H. Yoon; Sonya  A. Widen; Melissa  M. Wilson; Jennifer  C. Hocking; Andrew  J. Waskiewicz

doi:10.3791/59259

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

5.2K views

⸱

DOI:

10.3791/59259

⸱

March 27th, 2019

Kevin H. Yoon¹ , Sonya A. Widen¹^,² , Melissa M. Wilson¹ , Jennifer C. Hocking²^,³^,⁴^,⁵ , Andrew J. Waskiewicz¹^,²^,⁶

¹Department of Biological Sciences, University of Alberta, ²Women & Children's Health Research Institute, University of Alberta, ³Division of Anatomy, Department of Surgery, University of Alberta, ⁴Department of Cell Biology, University of Alberta, ⁵Department of Medical Genetics, University of Alberta, ⁶Neuroscience and Mental Health Research Institute, University of Alberta

Summary

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Abstract

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter's needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Introduction

The formation of the vertebrate eye is a highly conserved process in which carefully orchestrated intercellular signaling pathways establish tissue types and specify regional identity¹. Perturbations to early eye morphogenesis result in profound defects to the architecture of the eye and are frequently blinding². One such disease results from the failure to close the choroid ocular fissure in the ventral side of the optic cup³. This disorder, known as ocular coloboma, is estimated to occur in 1 out of 4-5000 live births and cause 3-11% of pediatric blindness, commonly manifesting as a keyhole-like....