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

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.

Ocular coloboma: Genetic variants reveal a dynamic model of eye development

“…There have been rare cases of patients presenting with both persistent fetal vasculature and coloboma, suggesting there might be a shared etiology or a cause‐and‐effect relationship between these two processes (Graziano et al, 2017; Ranchod et al, 2010; Rothfield et al, 2019; Takkar, Chandra, Kumar, & Agrawal, 2016; Weiner & Nudleman, 2019). The lobes of the choroid fissure and the developing hyaloid vasculature come into contact prior to closure of the fissure, suggesting that the hyaloid vessels may act as a scaffold that is necessary for choroid fissure closure (Eckert, Knickmeyer, & Heermann, 2020). Proper maintenance and development of the hyaloid vasculature is essential for choroid fissure closure and disrupting the hyaloid vessel can lead to disrupted choroid fissure closure (James et al, 2016; Weiss, Kaufman, Michaeli, & Inbal, 2012).…”

Closing the Gap: Mechanisms of Epithelial Fusion During Optic Fissure Closure

“…In zebrafish, the use of fluorescent reporter systems coupled to live-imaging confocal microscopy has been extremely powerful to determine specific cellular behaviors during OFC ( Gestri et al, 2018 ; Eckert et al, 2020 ). Two new studies have recently established chicken embryos ( Figure 2C ) as models for OFC research, both with particular focus on fusion events ( Bernstein et al, 2018 ; Hardy et al, 2019 ).…”

“…Cell mixing and fusion of the epithelial layers appears to lag several hours behind BM removal in zebrafish ( James et al, 2016 ). Recently, fusion was shown to occur closer to the optic nerve head in the proximal aspect of this axis ( Eckert et al, 2020 ). The length of the zebrafish fissure is approximately 60 μm and its closure is largely completed by 48 hpf, with the distal-most region being the last region to fuse by 56 hpf ( Figure 2B ).…”

“…These cells had flattened morphology just prior to fusion, consistent with RPE cell morphology, although they were not pigmented. These “pioneer cells” as the authors named them, became more cuboid before contact ( Eckert et al, 2020 ) then displayed amorphous elongated shapes with protrusions and overt polarization toward the edges of the fissure margin ( Gestri et al, 2018 ). Molecular analyses are still ongoing to precisely determine the characteristics and identity of these cells ( Figure 3A ), however, cell labeling and live imaging revealed they were negative for the NR progenitor reporter Rx2:GFP before and during fusion, but then gained Rx2:GFP expression in the fused margin ( Eckert et al, 2020 ).…”

“…However, it also indicates that pioneer cells could be a separate novel cell type within the OF. Indeed, the RPE marker dopachrome tautomerase ( dct ) was not observed in these cells ( Eckert et al, 2020 ). Although Netrin1a and integrina5 have been observed in this location in zebrafish ( Lupo et al, 2011 ) and this is a known region of localized PAX2 expression, it will be important to find specific markers for pioneer cells that can be used to perform robust characterization of their contributions to fusion (are they essential?)…”