In Vivo Analysis of Optic Fissure Fusion in Zebrafish: Pioneer Cells, Basal Lamina, Hyaloid Vessels, and How Fissure Fusion is Affected by BMP.

Priska Eckert,Max D Knickmeyer,Stephan Heermann

doi:10.3390/ijms21082760

Priska Eckert, Max D Knickmeyer + Show 1 more

Open Access

https://doi.org/10.3390/ijms21082760

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Abstract

Colobomata, persistent optic fissures, frequently cause congenital blindness. Here, we focused on optic fissure fusion using in vivo time-lapse imaging in zebrafish. We identified the fusion initiating cells, which we termed “pioneer cells.” Based on morphology, localization, and downregulation of the neuroretinal (NR) precursor marker rx2, these cells could be considered as retinal pigment epithelial (RPE) progenitors. Notably, pioneer cells regain rx2 expression and integrate into the NR after fusion, indicating that they do not belong to the pool of RPE progenitors, supported by the lack of RPE marker expression in pioneer cells. They establish the first cellular contact between the margins in the proximal fissure region and separate the hyaloid artery and vein. After initiation, the fusion site is progressing distally, increasing the distance between the hyaloid artery and vein. A timed BMP (Bone Morphogenetic Protein) induction, resulting in coloboma, did not alter the morphology of the fissure margins, but it did affect the expression of NR and RPE markers within the margins. In addition, it resulted in a persisting basal lamina and persisting remnants of periocular mesenchyme and hyaloid vasculature within the fissure, supporting the necessity of BMP antagonism within the fissure margins. The hampered fissure fusion had severe effects on the vasculature of the eye.

Highlights

During eye development in vertebrates, during optic cup morphogenesis, a fissure forms in the ventral part of the optic cup, termed optic fissure or choroid fissure [1]
We observed that the first cellular contact in between the fissure margins was established by cells with flattened morphology and a markedly reduced green fluorescence, driven by the aforementioned rx2 cis regulatory element [21] (Figure 1B,B’,C,C’, supplemental movie file 1, Figure S1A,A’,B,B’)
These findings indicate that the pioneer cells, even though they showed retinal pigment epithelial (RPE) progenitor features at the onset of contact formation, might not belong to the pool of RPE precursors but constitute a separate cell type that can gain NR progenitor features during fusion

Summary

Introduction

During eye development in vertebrates, during optic cup morphogenesis, a fissure forms in the ventral part of the optic cup, termed optic fissure or choroid fissure [1]. This fissure is a physiological (but transient) gap [2,3] and is important during a specific period of development for the entry of periocular mesenchymal cells (POMs) and POM-derived embryonic vasculature (hyaloid vessels) [4]. To some environmental and dietary factors, many genes have been linked to coloboma formation [7], resulting in a coloboma gene network [8,9] which recently was updated [6]. It consists of many signaling pathway components, e.g., of Wnt [10,11], fibroblast growth factor (FGF) [12,13], retinoic acid (RA) [14,15], Hippo [16], sonic hedgehog (Shh) [17], transforming growth factor β (TGFβ), and bone morphogenetic protein (BMP) [18,19]

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