Shi X et al. (2006), Zebrafish foxe3: roles in ocular lens morphogen...

XB-ART-34448

Mech Dev 2006 Oct 01;12310:761-82. doi: 10.1016/j.mod.2006.07.004.

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Zebrafish foxe3: roles in ocular lens morphogenesis through interaction with pitx3.

Shi X , Luo Y , Howley S , Dzialo A , Foley S , Hyde DR , Vihtelic TS .

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Foxe3 is a winged helix/forkhead domain transcription factor necessary for mammalian and amphibian lens development. Human FOXE3 mutations cause anterior segment dysgenesis and cataracts. The zebrafish foxe3 cDNA was PCR amplified from 24 h post-fertilization (hpf) embryo cDNA. The zebrafish foxe3 gene consists of a single exon on chromosome 8 and encodes a 422 amino acid protein. This protein possesses 44% and 67% amino acid identity with the human FOXE3 and Xenopus FoxE4 proteins, respectively. A polyclonal antiserum was generated against a bacterial fusion protein containing the Foxe3 carboxyl terminus. The purified antiserum detects zebrafish Foxe3 on immunoblots, in embryo wholemounts, and frozen tissue sections. The zebrafish Foxe3 protein is first detected in the lens at 31hpf and is restricted to the nucleated cell population, including the epithelial and elongating fiber cells. Knockdown of Foxe3 protein using an antisense morpholino results in small lenses with multilayered epithelial cells and fiber cell dysmorphogenesis. The morphants posses normal retinas, although retinal cell proteins, including rhodopsin, are abnormally expressed in the morphant lens tissue. Functional interactions between foxe3 and pitx3 during lens development were assessed by RT-PCR and comparison of Foxe3 and Pitx3 protein expression in both foxe3 and pitx3 morphants. Immunoblots and immunohistochemistry reveal Pitx3 is expressed in the foxe3 morphant lens, while Pitx3 knockdown results in the elimination of Foxe3 expression. These data demonstrate that Foxe3 is necessary for lens development in zebrafish and that foxe3 lies genetically downstream of pitx3 in a zebrafish lens development pathway.

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Species referenced: Xenopus
Genes referenced: actl6a foxe3 pitx3 rho

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Fig. 2. Temporal and spatial expression of the foxe3 gene. (A) RT-PCR demonstrates the temporal expression of foxe3 during embryonic development. A low level of maternal foxe3 transcript is detected (2 hpf) and foxe3 expression levels increase at 14–18 hpf. In addition, foxe3 transcription is detected in adult lens and brain. As a control, β-actin amplification is shown in the lower panel. (B–D) Whole-mount in situ hybridization demonstrates foxe3 spatial expression at 24 and 72 hpf. Expression of foxe3 is largely restricted to the eye (panel B, arrow) with highest transcript levels detected in the lens epithelial cells at 24 and 72 hpf (arrows in panels C and D, respectively). Restricted expression within the brain is also detected at 24 hpf (panel C, arrowheads).

Fig. 13. Immunolocalization of Pitx3 and Foxe3 in foxe3 and pitx3 morphant eye tissues. The Pitx3 and Foxe3 proteins were immunolocalized in frozen sections of either foxe3 or pitx3 morphant eyes and the relevant mismatch control morpholino-injected embryos (mmControl). Pitx3 is detected in the lens of foxe3 mmControl embryos at 3, 5 and 7 dpf (panels A, E and I, respectively), and also in the foxe3 morphant lens at these same time points (panels B, F and J). The Pitx3 spatial expression pattern is disrupted in the foxe3 morphants. A wild-type Foxe3 expression pattern is displayed in the pitx3 mmControl embryos at 3, 5 and 7 dpf (panels C, G and K, respectively). In contrast, Foxe3 protein is not detected in the pitx3 morphants at 3, 5 and 7 dpf (panels D, H and L, respectively; arrows delineate the lens tissue). The scale bar in panel A (50 μm) applies to all the panels. Abbreviations: PL, photoreceptor layer; INL, inner nuclear layer; GCL, ganglion cell layer.