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.

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Notes/Citation Information

Published in Frontiers in Cell and Developmental Biology, v. 8, article 379, p. 1-16.

© 2020 Van Der Meulen, Vöcking, Weaver, Meshram and Famulski.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Funding Information

This work was supported by the NIH-NEI grant EY027805-01. MW was supported by the Lyman T. Johnson Scholarship from the University of Kentucky. OV was supported by the Knights Templar Eye Foundation Career Starter Grant.

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The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2020.00379/full#supplementary-material

The Supplementary Material is also available as the additional files listed at the end of this record.

table 1.xlsx (12 kB)
Table S1: WISH Primer Sequences. mRNA forward and reverse primer sequences for all POM and NCC-related genes.

table 2.xlsx (106 kB)
Table S2: scRNA sequencing aggregation gene list. Distribution of all gene expression analyzed during aggregation analysis.

video 1.mp4 (2603 kB)
Video S1: Foxc1b:GFP 4D imaging (24–28 hpf).

video 2.mp4 (2006 kB)
Video S2: Foxd3:GFP 4D imaging (24–48 hpf).

video 3.mp4 (1624 kB)
Video S3: Pitx2:GFP 4D imaging (22–46 hpf).

video 4.mp4 (1443 kB)
Video S4: Lmx1b.1:GFP 4D imaging (24–48 hpf).

video 5.mp4 (10097 kB)
Video S5: Sox10:RFP 4D imaging (23–47 hpf).

video 6.mp4 (2424 kB)
Video S6: Foxc1b:GFP Tracking analysis.

video 7.mp4 (1837 kB)
Video S7: Foxd3:GFP Tracking analysis.

video 8.mp4 (922 kB)
Video S8: Pitx2:GFP Tracking analysis.

video 9.avi (2731 kB)
Video S9: Lmx1b.1 Tracking analysis.

video 10.avi (1773 kB)
Video S10: Sox10:RFP Tracking analysis.

image 1.tif (242 kB)
Figure S1: Schematic representation of AS colonization in a zebrafish embryo.

image 2.tif (4482 kB)
Figure S2: Anterior segment ASM heterogeneity for eya2 expression.

image 3.tif (2060 kB)
Figure S3: PH3 proliferation staining assay.

image 4.tif (424 kB)
Figure S4: cDNA library-based t-SNE and UMAP clusters.

image 5.tif (899 kB)
Figure S5: PANTHER Gene Ontology for 48 hpf ASM clusters.