Spatiotemporal Characterization of Anterior Segment Mesenchyme Heterogeneity During Zebrafish Ocular Anterior Segment Development

Abstract

Assembly of the ocular anterior segment (AS) is a critical event during development of the vertebrate visual system. Failure in this process leads to anterior segment dysgenesis (ASD), which is characterized by congenital blindness and predisposition to glaucoma. The anterior segment is largely formed via a neural crest-derived population, the Periocular Mesenchyme (POM). In this study, we aimed to characterize POM behaviors and transcriptional identities during early establishment of the zebrafish AS. Two-color fluorescent in situ hybridization suggested that early AS associated POM comprise of a heterogenous population. In vivo and time-course imaging analysis of POM distribution and migratory dynamics analyzed using transgenic zebrafish embryos (Tg[foxc1b:GFP], Tg[foxd3:GFP], Tg[pitx2:GFP], Tg[lmx1b.1:GFP], and Tg[sox10:GFP]) revealed unique AS distribution and migratory behavior among the reporter lines. Based on fixed timepoint and real-time analysis of POM cell behavior a comprehensive model for colonization of the zebrafish AS was assembled. Furthermore, we generated single cell transcriptomic profiles (scRNA) from our POM reporter lines and characterized unique subpopulation expression patterns. Based on scRNA clustering analysis we observed cluster overlap between neural crest associated (sox10/foxd3), POM (pitx2) and finally AS specified cells (lmx1b, and foxc1b). scRNA clustering also revealed several novel markers potentially associated with AS development and/or function including lum, fmoda, adcyap1b, tgfbi, and hmng2. Taken together, our data indicates that AS-associated POM, or Anterior Segment Mesenchyme (ASM), is not homogeneous but rather comprised of several subpopulations with differing colonization patterns, migration behavior, and transcriptomic profiles.

Mechanistic Insights into Axenfeld–Rieger Syndrome from Zebrafish foxc1 and pitx2 Mutants

“…At early stages of development, zebrafish express foxc1a and foxc1b in overlapping domains in neural crest cells [ 4 , 20 ] ( Figure 1 G, ( foxc1a )) as well as the lateral plate mesoderm (LPM) [ 21 ]. Neural crest cells contribute to the periocular mesenchyme (POM)—a set of cells that are required for closure of the optic fissure and development of the ocular anterior segment [ 22 ]. Neural crest cells also populate the central nervous system, craniofacial skeleton, smooth muscle cells of the cerebral vasculature, as well as the cardiac outflow tract and heart valves [ 23 , 24 , 25 ].…”

“…The expression of pitx2a is found in partially overlapping domains with foxc1a and foxc1b in developing zebrafish embryos. As early as 24 h post fertilization (hpf), pitx2 expression is observed in the periocular mesenchyme [ 22 ] ( Figure 2 A), providing an opportunity for co-regulation of gene expression with Foxc1a and Foxc1b. Like foxc1a and foxc1b , pitx2 is expressed in the neural crest derived tissues of the pharyngeal arches that contribute to the craniofacial skeleton [ 30 ].…”

The Ocular Neural Crest: Specification, Migration, and Then What?

“…Sox10-positive cells migrate early after optic cup formation and preliminary fate mapping studies show minimal contributions to the adult eye (unpublished data). Foxd3-positive NC cells migrate into the anterior segment via two pathways: (1) adjacent to the hyaloid vasculature within the optic fissure and (2) between the surface ectoderm and the optic cup ( Mork and Crump, 2015 ; Williams and Bohnsack, 2017 ; Van Der Meulen et al, 2020 ; Figure 3 ). Foxd3-positive cells have a continued presence within the corneal endothelium and iris in zebrafish larvae and young juveniles.…”

“…With the loss of early markers, such as Sox10 and Crestin, NC cells adopt the expression of other transcription factors, namely Pitx2, Foxc1, Lmx1b, and Eya2 ( Van Der Meulen et al, 2020 ). Further, signaling molecules, such as retinoic acid, and interactions between NC cells and the adjacent ocular tissues have continued effects on the ocular migration and end differentiation of NC cells.…”

Genetics Underlying the Interactions between Neural Crest Cells and Eye Development

“…Crestin , a well-defined marker for migratory neural crest cells is expressed in the dorsal, but not ventral periocular mesenchyme [ 173 ]. In zebrafish, while there is initially coexpression of sox10 and foxd3 during early migration into the pharyngeal arches and periocular mesenchyme, the timing of the down regulation of these transcription factors varies depending on the triggering of terminal differentiation [ 11 , 174 ]. During this process, the expression of additional transcription factors including pitx2 , foxc1 , eya2 , and lmx1b is upregulated in cranial neural crest cells in order to regulate migration to final destinations and differentiation ( Figure 4 ) [ 174 ].…”