notum1, acting downstream of pitx2, is essential for proper eye and craniofacial development

Abstract

Axenfeld-Rieger syndrome (ARS) is a rare autosomal dominant developmental disorder characterized by ocular anterior chamber anomalies with an increased risk of glaucoma and systemic defects. Mutations in the transcription factor PITX2 were the first identified genetic cause of ARS. Despite the developmental importance of PITX2 and its role in ARS, the pathways downstream of PITX2 have yet to be fully characterized. Comparative transcriptome analyses involving pitx2-enriched cell populations isolated via fluorescence activated cell sorting of tissues expressing (Tg(-2.6pitx2-CE4:GFP)) reporter in wild-type and pitx2 M64* mutant zebrafish embryos identified the highly down-regulated target notum1b, an ortholog of human NOTUM encoding a secreted carboxylesterase that cleaves a necessary palmitoleate moiety from WNT proteins.Further experiments confirmed a decrease in notum1b and identified down-regulation of another NOTUM ortholog, notum1a, in the developing mutant eye. CRISPR-generated permanent double knockout zebrafish lines of notum1b and notum1a, notum1-/-, displayed defects in craniofacial and ocular development, including corneal defects, small lenses, increased sizes of the anterior and posterior chambers, and anomalies in teeth development. Analysis of head transcriptome of notum1-/-zebrafish in comparison to wild-type predicted an upregulation of the WNT pathway. We present NOTUM/notum1 as an important factor in ocular and craniofacial development and a novel downstream member of the PITX2/pitx2 pathway.

Deletions of distant regulatory sequences upstream of zebrafish pitx2 result in a range of ocular phenotypes

“…They found that Pitx2 is expressed at e10.5 but is then quickly lost as development continues, suggesting that WNT/β-catenin signaling is essential for the maintenance of Pitx2 expression after activation by RA signaling. Interestingly, two WNT antagonists, Dkk2 and notum , were shown to be downstream of Pitx2/pitx2 with Dkk2 demonstrated to be a direct target (Gage et al 2008; Hendee et al 2019). Currently available data are not sufficient to fully understand the transcriptional landscape governing the spatio-temporal expression pattern of pitx2/pitx2 , therefore additional studies are necessary to understand what regulates its expression.…”