Pitx2 Deficiency Results in Abnormal Ocular and Craniofacial Development in Zebrafish

Yi Liu,Elena V Semina,Domingos Henrique

doi:10.1371/journal.pone.0030896

Yi Liu, Elena V Semina + Show 1 more

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https://doi.org/10.1371/journal.pone.0030896

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Abstract

Human PITX2 mutations are associated with Axenfeld-Rieger syndrome, an autosomal-dominant developmental disorder that involves ocular anterior segment defects, dental hypoplasia, craniofacial dysmorphism and umbilical abnormalities. Characterization of the PITX2 pathway and identification of the mechanisms underlying the anomalies associated with PITX2 deficiency is important for better understanding of normal development and disease; studies of pitx2 function in animal models can facilitate these analyses. A knockdown of pitx2 in zebrafish was generated using a morpholino that targeted all known alternative transcripts of the pitx2 gene; morphant embryos generated with the pitx2ex4/5 splicing-blocking oligomer produced abnormal transcripts predicted to encode truncated pitx2 proteins lacking the third (recognition) helix of the DNA-binding homeodomain. The morphological phenotype of pitx2ex4/5 morphants included small head and eyes, jaw abnormalities and pericardial edema; lethality was observed at ∼6–8-dpf. Cartilage staining revealed a reduction in size and an abnormal shape/position of the elements of the mandibular and hyoid pharyngeal arches; the ceratobranchial arches were also decreased in size. Histological and marker analyses of the misshapen eyes of the pitx2ex4/5 morphants identified anterior segment dysgenesis and disordered hyaloid vasculature. In summary, we demonstrate that pitx2 is essential for proper eye and craniofacial development in zebrafish and, therefore, that PITX2/pitx2 function is conserved in vertebrates.

Highlights

IntroductionThe majority of PITX2 mutations are located in the homeobox region encoding the homeodomain and affect all known PITX2 isoforms (see below)
PITX2 is a member of the bicoid-like homeodomain transcription factor family which, when mutated, is responsible for AxenfeldRieger syndrome (MIM ID #180500), an autosomal-dominant developmental disorder characterized by ocular anterior chamber anomalies, increased risk for glaucoma, dental hypoplasia, craniofacial dysmorphism and umbilical region abnormalities [1].The majority of PITX2 mutations are located in the homeobox region encoding the homeodomain and affect all known PITX2 isoforms
RT-PCR analysis of mRNA extracted from pitx2ex4/5 morphants identified an acute reduction or a complete absence of normal pitx2 transcripts along with the presence of an aberrant PCR product at 24–48-hpf (Figure 1B); the quantity of normal transcript continued to be significantly diminished in 72– 96-hpf pitx2ex4/5 morphants and reached normal levels in 120-hpf embryos (Figure 1B)

Summary

Introduction

The majority of PITX2 mutations are located in the homeobox region encoding the homeodomain and affect all known PITX2 isoforms (see below). These mutations typically generate a nullallele, which supports PITX2 haploinsufficiency as a mechanism for Axenfeld-Rieger syndrome, consistent with reports of gene deletion in some patients; PITX2 mutations that appear to retain limited wild-type activities are usually associated with milder phenotypes [2]. Several PITX2 isoforms have been reported with four transcripts, PITX2A-D, identified in humans [12], three, Pitx2a– c, in mice and frogs [13] and two, pitx2a and pitx2c, described in chickens [14], zebrafish [15] and even ascidians [16]; the isoforms have different N-terminal sequences but share exons encoding for the homeodomain and C-terminal region. Some variations were observed, the Pitx isoforms demonstrate largely overlapping expression patterns [13,14,15,17]; functional assays demonstrate comparable DNA-binding and transactivation activities for the main isoforms with minor differences revealed under certain conditions [12,18]

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