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.
ABSTRACT
Vertebrate retinal development requires timely and precise fusion of the optic fissure (OF). Failure of this event leads to congenital vision impairment in the form of coloboma. Recent studies have suggested hyaloid vasculature to be involved in OF fusion. In order to examine this link, we analyzed OF fusion and hyaloid vasculogenesis in the zebrafish pax2a noi mutant line. We first determined that pax2a-/- embryos fail to accumulate F-actin in the OF prior to basement membrane (BM) degradation. Furthermore, using 3D and live imaging we observed reduced OF hyaloid vascularization in pax2a-/- embryos. When examining the connection between pax2a loss of function and hyaloid vasculature, we observed significant reduction of talin1 expression, a regulator of hyaloid vasculature. In addition, cranial VEGF expression was found to be reduced in pax2a-/- embryos. Pharmacological inhibition of VEGF signaling phenocopied the pax2a-/- vasculature, F-actin and BM degradation phenotypes. Lastly, we determined that OF associated hyaloid vasculature is a source of mmp2, mmp14a and mmp14b expression and showed that mmp2 is functionally necessary for degradation of OF BM. Taken together we propose a pax2a driven mechanism that ensures proper and timely hyaloid vasculature invasion of the OF in order to facilitate availability of the BM remodeler mmp2.
