Zebrafish pitx3 is necessary for normal lens and retinal development

Abstract

The human PITX3 gene encodes a bicoid-like homeodomain transcription factor associated with a variety of congenital ocular conditions, including anterior segment dysgenesis, Peter's anomaly, and cataracts. We identified a zebrafish pitx3 gene encoding a protein (Pitx3) that possesses 63% amino acid identity with human PITX3. The zebrafish pitx3 gene encompasses approximately 16.5kb on chromosome 13 and consists of four exons, which is similar to the genomic organization of other pitx genes. Expression of the zebrafish pitx3 gene was studied by in situ mRNA hybridization and RT-PCR. The pitx3 transcripts were detected throughout development with the greatest level of expression occurring in the developing lens and brain at 24hpf. In adults, the highest expression was detected in the eye. Morpholinos were used to knockdown expression of the Pitx3 protein and a control morpholino that contains five mismatched bases was used to confirm the specificity of the phenotypes. The morphants had small eyes, misshapen heads and reduced jaws and fins relative to controls. The morphants exhibited abnormalities in lens development and their retinas contained pyknotic nuclei accompanied by a reduction in the number of cells in different neuronal classes. This suggests the lens is required for retinal development or Pitx3 has an unexpected role in retinal cell differentiation or survival. These results demonstrate zebrafish pitx3 represents a true ortholog of the human PITX3 gene and the general function of the Pitx3 protein in lens development is conserved between mammals and the teleost fish.

laminin alpha 1gene is essential for normal lens development in zebrafish

“…Genes responsible for the aphakia and dysgenetic lens phenotypes have been identified as transcription factors, Pitx3 and Foxe3 ; both genes were also shown to be involved in human ocular disorders involving abnormal lens, iris and corneal development [54,55] and zebrafish pitx3 -morphants displayed lens degeneration similar to mammals [70]. Defects in the basement membrane and/or extracellular matrix were reported in aphakia and lens aplasia mutants [69,71] indicating a possible connection with the laminin and/or other extracellular matrix molecule pathway(s) that needs to be further investigated.…”

“…PCR products were generated using specific oligos, PfuUltra high-fidelity DNA polymerase (Stratagene, La Jolla, CA) and standard conditions described elsewhere [70]. The PCR products were separated by electrophoresis in 1% agarose gel, cloned into a pCRII-TOPO vector (Invitrogen, Carlsbad, CA) and subjected to DNA sequencing using the ABI PRISM 373 DNA Sequencer.…”

Zebrafish dou yan mutation causes patterning defects and extensive cell death in the retina

“…Previous mutagenesis screens and reverse genetic approaches have identified several genes that are important in controlling the size of zebrafish eye: Rab11-FIP4, Mcm5, PITX3, small heart, no tectal neuron, and dazed (Matsuda and Mishina, 2004;Yuan and Joseph, 2004;Babb et al, 2005;Perkins et al, 2005;Shi et al, 2005;Ryu et al, 2005;Muto et al, 2006). These genes map to chromosome regions different from the one in which the dou yan gene resides.…”

“…Furthermore, in addition to the small eye phenotype, malfunction of these gene products also causes other defects. For example, disruption of Rab11-FIP4 and Mcm5 genes leads to extensive cell death not only in the retina but also in the brain (Ryu et al, 2005;Muto et al, 2006); the small heart mutation causes malformation of the heart and a curled body axis (Yuan and Joseph, 2004); disruption of the PITX3 gene function, which is required for lens development, likely has an indirect effect on retinal development as a consequence of disrupted interactions between lens and retina (Yamamoto and Jeffery, 2000;Shi et al, 2005); in no tectal neuron, the small eye phenotype does not appear Fig. 9.…”

“…Some of these genes are involved in cell proliferation, such as Rb, Mcm5, and XOptx2 (Friend et al, 1987;Zuber et al, 1999;Ryu et al, 2005;Muto et al, 2006). Other genes affect cell differentiation, such as shh, pitx3, and dzd (Neumann and Nuesslein-Volhard, 2000;Wang et al, 2005;Shi et al, 2005;Perkins et al, 2005). Still others are necessary for the survival of retinal cells at early development stages, such as mikre oko, oval, and elipsa, or for the survival of retinal cells in adults, such as pde6b and TULPs (Pittler and Baehr, 1991;Hagstrom et al, 1998;Reme et al, 1998;Malicki, 2001, 2002).…”