Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs

Abstract

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2−/− knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFβ) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling—independent of Pitx2—controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis.

Spatiotemporal characterization of periocular mesenchyme heterogeneity during anterior segment development

“…The most common mutations seen in ASD patients involve the transcription factor pitx2 (Paired-like homeodomain) (11), as well as foxC1 (Forkhead Box C1) (10, 13–16). Loss of function of either pitx2 or foxc1 has been shown to result in ASD phenotypes in mice and zebrafish (11, 13–18).…”

“…The most common mutations seen in ASD patients involve the transcription factor pitx2 (Paired-like homeodomain) (11), as well as foxC1 (Forkhead Box C1) (10, 13–16). Loss of function of either pitx2 or foxc1 has been shown to result in ASD phenotypes in mice and zebrafish (11, 13–18). Pitx2 in particular has been associated with the survival and migration of NCCs, as well as the development of the optic stalk, establishment of angiogenic privilege within the cornea, and craniofacial development (10, 11, 14, 17–21).…”

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

“…Ocular features have a later onset and include corneal anomalies in all three layers and disproportionally small lens resulting in myopia. These defects overlap features associated with pitx2/Pitx2 deficiency including loss of corneal integrity, jaw and dental anomalies (Gage et al, 1999;Hendee et al, 2018;Ji, Buel, & Amack, 2016;Li et al, 2014) as well as Dkk2 deficiency, which also displays compromised corneal identity (Gage et al, 2008;Mukhopadhyay et al, 2006). The presented data suggest that PITX2/pitx2 acts on multiple targets (NOTUM/notum1, DKK2/dkk2) to negatively affect the WNT pathway during anterior segment development and their combined function is required for the proper eye formation.…”

Left–Right Patterning: Breaking Symmetry to Asymmetric Morphogenesis

“…However, some morphological asymmetries are not affected by Pitx2 loss including heart looping, axial rotation, and stomach sidedness, suggesting that PITX2-independent signals downstream of Nodal are important for some aspects of asymmetric morphogenesis. Such signals may be even more relevant in zebrafish, since loss of pitx2 in this model does not affect organ asymmetry [15]. …”