PITX2 deficiency and associated human disease: insights from the zebrafish model

Abstract

The PITX2 (paired-like homeodomain 2) gene encodes a bicoid-like homeodomain transcription factor linked with several human disorders. The main associated congenital phenotype is Axenfeld-Rieger syndrome, type 1, an autosomal dominant condition characterized by variable defects in the anterior segment of the eye, an increased risk of glaucoma, craniofacial dysmorphism and dental and umbilical anomalies; in addition to this, one report implicated PITX2 in ring dermoid of the cornea and a few others described cardiac phenotypes. We report three novel PITX2 mutations—c.271C > T, p.(Arg91Trp); c.259T > C, p.(Phe87Leu); and c.356delA, p.(Gln119Argfs*36)—identified in independent families with typical Axenfeld-Rieger syndrome characteristics and some unusual features such as corneal guttata, Wolf-Parkinson-White syndrome, and hyperextensibility. To gain further insight into the diverse roles of PITX2/pitx2 in vertebrate development, we generated various genetic lesions in the pitx2 gene via TALEN-mediated genome editing. Affected homozygous zebrafish demonstrated congenital defects consistent with the range of PITX2-associated human phenotypes: abnormal development of the cornea, iris and iridocorneal angle; corneal dermoids; and craniofacial dysmorphism. In addition, via comparison of pitx2M64* and wild-type embryonic ocular transcriptomes we defined molecular changes associated with pitx2 deficiency, thereby implicating processes potentially underlying disease pathology. This analysis identified numerous affected factors including several members of the Wnt pathway and collagen types I and V gene families. These data further support the link between PITX2 and the WNT pathway and suggest a new role in regulation of collagen gene expression during development.

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

“…Additionally, the pharyngeal arch cartilages of the lower jaw exhibited structural and positional defects. These features emulated several key features observed in ARS as well as rare phenotypes connected with PITX2, namely ring dermoid of the cornea (Hendee et al, 2018).…”

“…Differentially expressed targets were defined as having a difference in mean log(2) values > +1 or < -1 and a p-value < 0.05. Zebrafish-specific transcripts were annotated with human orthologs as previously described (Hendee et al, 2018).…”

“…Hematoxylin and eosin (H&E) staining, Periodic acid Schiff (PAS) staining, and immunohistochemistry were performed on fresh slides per standard protocols as previously described (Hendee et al, 2018). H&E and PAS slides were imaged on a NanoZoomer digital slide scanner, and images were viewed with NDP.view2 viewing software (Hamamatsu, Hamamatsu City, Japan).…”

“…In situ hybridization (ISH) and RNAscope were performed as previously described (Hendee et al, 2018;Liu & Semina, 2012). Transcript-specific primers were utilized for contig mapping of notum1b as well as, along with DIG RNA labeling mix and T7 RNA polymerase (Roche Diagnostics Corporation, Indianapolis, IN), generation of one notum1a and four notum1b digoxigenin-labeled anti-sense RNA probes (notum1a: 623 bp; notum1b: 756, 1424, 763, and 770 bp) (Table S1).…”

“…In developing craniofacial structures, distal-less homeobox 2 (Dlx2) was shown to be downstream of Pitx2 within the dental epithelium during tooth development (Green et al, 2001). Transcriptome analysis of ocular tissues from our pitx2 M64* zebrafish line suggested several genes, namely collagens (e.g., I and V) and Wnts (e.g., 3 and 10a), as additional potential downstream targets of pitx2 (Hendee et al, 2018). Further dissection of the PITX2/pitx2 pathway through identification of its critical downstream targets is needed to better understand its role in embryonic development and human disease.…”