Kenyon KL et al. (1999), A novel fork head gene mediates early steps dur...

XB-ART-12107

Development 1999 Nov 01;12622:5107-16. doi: 10.1242/dev.126.22.5107.

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A novel fork head gene mediates early steps during Xenopus lens formation.

Kenyon KL , Moody SA , Jamrich M .

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Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike pax6, Xlens1 cannot induce the expression of the lens differentiation marker beta-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of gamma-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity.

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Species referenced: Xenopus
Genes referenced: cryba1 crybg3 crygdl.30 crygdl.43 foxe3 pax6 six3
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	Fig. 2. Expression of Xlens1 mRNA during development. (A-D) are anterior views of the head and (E-F) are lateral views. (A) Xlens1 is first detected at stage 13 in a narrow field across the anterior midline (arrow), separating the anterior neural plate (np) from ventral head epidermis (he). (B) At stage 15 Xlens1 expression has spread laterally to the anterior- lateral margins of the neural plate (np), reaching the limits of the trigeminal placode anlagen (arrow). (C) At stage 18 the midline expression of Xlens1 is depleted (arrow), and the lateral patches adjacent to the anterior neural tube (nt) are prominent. (D) At stage 22 Xlens1 expression is confined to the ectoderm overlying the optic vesicle. (E) At stage 28, Xlens1 expression is confined to the ectoderm overlying the optic cup. (F) At stage 34, only the lens vesicle expresses Xlens1. (G) Sagittal section through the midline band at stage 15. Xlens1- expressing cells are in the superficial (s) and deep (d) layers of the ectoderm, just anterior to the anterior rim (arrowhead) of the neural plate (np). Anterior, right; dorsal top. (H) Sagittal section through the midline at stage 18. Xlens1 expression is mostly depleted from the midline cells of the anterior ectodermal placode (ap), which will become the adenohypophyseal and olfactory placodes. A few Xlens1-expressing cells are still detected (white arrowhead) just anterior to the most rostral edge (black arrowhead) of the neural tube (nt). (I) Sagittal section through the lateral patch of Xlens1 expression in the same stage 18 embryo as in H. Xlens1 expression is mostly depleted from the placode (ap) anterior to the neural tube (nt), but is at high levels in both the superficial and deep (sensorial) layers of the ectoderm overlying the lateral aspects of the neural tube. Arrowhead delineates between the neural tube and the placode. (J) Transverse section through the optic vesicle (ov) at stage 22 embryo. Xlens1 expression is confined to the ectoderm overlying the vesicle. There is a sharp depletion of Xlens1 expression at dorsal and ventral points (arrowheads) where mesoderm intervenes between the optic vesicle and surface ectoderm. di, diencephalon. Dorsal, up; lateral, right. (K) Higher magnification of J, demonstrating that only the deep (d, sensorial) layer of cells in the PLE express Xlens1. Some cells (arrowheads) appear to have downregulated Xlens1 expression. s, superficial layer. (L) Transverse section of a stage 31/32 lens vesicle. Xlens1 is expressed only in the anterior epithelial cells of the lens (ae), adjacent to the surface ectoderm. The inner layer of the surface ectoderm (cornea) covering the lens vesicle also appears to express this gene (arrowhead). r, retina. (M) Transverse section of a stage 34 lens vesicle depicting both β-crystallin (blue) and Xlens1 (brown) expression. The lens fiber cells (lf) express β-crystallin, but do not express Xlens1. Magnification, 70(G-I), 75(J), 170(K), 250(L,μ).
	Fig. 4. Overexpression of Xlens1 causes thickening in lens ectoderm and suppresses differentiation of lens fibers. (A) Differential interference contrast image of the uninjected side showing the normal histological relationship between the lens vesicle (lv) and the surface ectoderm, which is composed of two thin layers of cells. r, retina. 190 (B) The injected side of the same embryo contains a smaller lens vesicle (lv) and a thickened lens ectoderm (le). r, retina. (C) Confocal image of a transverse section from stage 35. On the uninjected side (left) the expression of γ-crystallin protein (red) is comparable to that in normal, unperturbed embryos, with strong expression in the fibers of the lens vesicle (lv). On the injected side (right), γ-crystallin expression is confined to the fibers of a greatly reduced lens vesicle. The thickened lens ectoderm expresses high levels of Xlens1 (green). 190 (D,E) Cell nuclei stained with Hoechst. On control side (D), the ectoderm overlying the lens vesicle (lv) is 2 cell layers thick. On the injected side (E), the thickened ectoderm is 4-6 cell layers thick (between arrows). Magnification, 190(A-C), 240(D,ε).
	Fig. 5. In Xlens1-injected embryos, the thickened ectoderm expresses markers indicative of a PLE state of determination (A) In controls, pax6 is expressed in the PLE. ov, optic vesicle. (B) In controls, pax6 is expressed in the anterior lens epithelium of the lens vesicle. r, retina. (C) When Xlens1 is overexpressed, pax6 expression is expanded throughout the thickened ectoderm (arrows) overlying the retina in a pattern identical to control PLE (A). (D) In controls, six3- like is expressed in the deep layer of the PLE. (E) In controls, six3- like expression in the deep layers of lens ectoderm is downregulated when the lens placode begins to form (arrow). oc, optic cup. (F) When Xlens1 is overexpressed, six3-like expression is maintained in the deep layers of the thickened lens ectoderm (arrows), similar to control PLE (D). (G) In controls, γ-crystallin is first expressed throughout lens placode. It is not expressed in the PLE. (H) In controls, γ-crystallin is expressed throughout the lens vesicle. (I) When Xlens1 is overexpressed, γ-crystallin expression is absent from the thickened lens ectoderm (arrows), a pattern identical to control PLE (not shown). Magnification, 150(A,C,D,G), 165(H), 175(B), 190(E).
	Fig. 6. Overexpression of Xlens1 in selected regions of the anterior head of the embryo results in ectopic γ-crystallin protein expression. A duplicate set of confocal images acquired in a Z-series through 7 μm demonstrates that GFP-expression (A) and γ-crystallin expression (B) are in the same cells. In (C) the images are merged. Arrowhead in B indicates normal γ-crystallin staining in the native lens. 170
	Fig. 1. Structure and sequence of the Xlens1 cDNA. (A) Schematic representation of the Xlens1 cDNA (1474 bases), including putative translational start sites (vertical lines) 5¢ to the coding region (black box, 1093 bases), and the fork head domain within it (gray box, 303 bases). (B) The nucleotide sequence and translated coding region for Xlens1. The fork head domain is underlined and the polyadenylation signal site is bold. (C) Comparison of the amino acid sequence of Xlens1 and those members of the fork head family most closely related to Xlens1. Dots represent identity. RGKPPYSY is one of the conserved regions that indicate Xlens1 and its four closest relatives may represent a new subfamily in the class 8 group. H1, H2, H3 indicate the three a- helical regions. W1 and W2 indicate the two wing-like regions. GenBank accession number for Xlens1 is AF186464.
	Fig. 3. Animal cap assays of pax6 and Xlens1 injections. The horizontal axis denotes genes overexpressed in explants and the vertical axis genes tested for induction. Histone 4 (H4) demonstrates successful cDNA synthesis. con, control uninjected explants; we, whole embryo; -rt, minus reverse transcription product. (A) pax6 (200 pg) overexpression induces expression of Xlens1 in explants cultured until stages 22-24. This induction is detected at 28 cycles (lane 2) and there is an increase in amplified product at 30 cycles (lane 3) for the same amount of starting template, indicating that detection occurs within a linear range. Xlens1 is still present in the adult eye (lane 5). (B) Xlens1 does not induce expression of pax6 in explants cultured until stages 22-24 (30 cycles). Two doses of Xlens1 mRNA (10 pg, lane 2, and 100 pg, lane 3) were tested. (C) Xlens1 (200 pg) expression does not induce either pax6 or B-crystallin in explants cultured until stage 34 (30 cycles).