Investigation of Frizzled-5 during embryonic neural development in mouse

Abstract

Recent studies revealed that the Wnt receptor Frizzled-5 (Fzd5) is required for eye and retina development in zebrafish and Xenopus, however, its role during mammalian eye development is unknown. In the mouse embryo, Fzd5 is prominently expressed in the pituitary, distal optic vesicle, and optic stalk, then later in the progenitor zone of the developing retina. To elucidate the role of Fzd5 during eye development, we analyzed embryos with a germline disruption of the Fzd5 gene at E10.25, just before embryos die due to defects in yolk sac angiogenesis. We observed severe defects in optic cup morphogenesis and lens development. However, in embryos with conditional inactivation of Fzd5 using Six3-Cre, we observed no obvious early eye defects. Analysis of Axin2 mRNA expression and TCF/LEF-responsive reporter activation demonstrate that Fzd5 does not regulate the Wnt/beta-catenin pathway in the eye. Thus, the function of Fzd5 during eye development appears to be species-dependent.

Figure 1. Fzd5 expression pattern in ventral forebrain structures

Whole mount in situ hybridization of Fzd5 mRNA expression showing frontal (A, B), lateral (C) and ventral views (D) of the embryonic mouse head. (A) Fzd5 is expressed in the anterior neural plate at E8 (bracket, 6–8 somites; fg: foregut). (B) At E9, Fzd5 expression is detectable in the anterior forebrain (bracket). (C) At E9.5, Fzd5 is expressed in the optic vesicle (ov) and optic stalk as well as in the olfactory placode (white arrowhead) and lung buds (black arrowhead). (D) By E10.5, Fzd5 is expressed in the ventral forebrain, Rathke’s pouch (rp) and infundibulum (inf). Fzd5 expression was also detected in the olfactory placode (black arrowheads) and along the optic stalks (white arrowheads). E: embryonic day, Lg: lung

Figure 2. Fzd5 expression in the embryonic and perinatal mouse eye

(A) Fzd5 mRNA is expressed in the presumptive neural retina (arrowhead) and optic stalk of the optic vesicle (ov) at E9.5 but not in the future RPE or overlying lens ectoderm. (B) Fzd5 expression is maintained in the neural retina and optic stalk at E11.5. (C) By E13.5, Fzd5 expression is decreased in the presumptive ganglion cell layer (GCL), but maintained in the progenitor zone. In addition, some Fzd5 expression is present in the dorsal optic stalk (arrowhead). (D) Subsequently, Fzd5 becomes restricted to the neuroblastic layer (NBL) in the P1 retina and is downregulated in the future outer nuclear layer (ONL). P: postnatal day. All scale bars are 100μm.

Figure 3. Expression of Fzd5 mRNA in the developing pituitary

(A) During pituitary organogenesis, Fzd5 is expressed in the anterior neural plate at E8 (anr; sagittal section; 6–8 somites). At E9, Fzd5 expression is detectable in the ventral forebrain (sagittal section in B, coronal section in C) as well as the oral ectoderm (B, arrow). By E10.5, Fzd5 is expressed in the ventral forebrain (sagittal section in D, coronal section in E), oral ectoderm (arrow in D) and Rathke’s pouch (arrow in E). Fzd5 expression was also detected in the olfactory placode (F; black arrowhead, coronal section).

Figure 4. Fzd5 does not appear to inhibit or activate the canonical pathway

Control (A) or Fzd5−/− embryos (B) carrying the TOPGAL transgene were analyzed by Xgal staining. Activation of the TOPGAL reporter is restricted to the dorsal optic vesicle in Fzd5−/− embryos at E9.5, similar to control embryos (A, B; arrowheads). Expression of Axin2 at 30 somites is restricted to the dorsal optic cup (arrowhead in C).

Figure 5. Fzd5−/− embryos fail to develop an optic cup

Morphology of control (wildtypes, A, C) and Fzd5−/− embryos (B, D) was analyzed by hematoxylin and eosin staining using frontal sections of paraffin embedded tissue, dorsal is toward the top in all panels. Fzd5−/− and control embryos develop a morphologically normal optic vesicle at E9.5 (compare A and B). By E10.75, the Fzd5−/− eye has failed to form the bilayered optic cup, unlike the control littermates (compare C and D; arrowhead). The lens placode does invaginate to some extent in Fzd5−/− embryos (inset in D; arrow). Control (E) or mutant embryos (F) were analyzed for Tunel labeling (green; DAPI: red). At E10 (30 somites), Fzd5−/− embryos showed increased Tunel labeling in the presumptive neural retina compared with controls (F; arrow). To determine effects on proliferation, BrdU incorporation was determined at E10 (G). The proportion of BrdU positive cells was significantly reduced in the distal (25.03 ± 14.14) and ventral optic vesicle (33.1 ± 12.67) in Fzd5−/− embryos in comparison to controls (distal: 53.35 ± 5.67; ventral: 50.80 ± 5.10; n = 3 embryos). In contrast, proliferation did not significantly change in the dorsal diencephalon where Fzd5 is not expressed (55.27 ± 5.27 in controls versus 52.62 ± 5.05 in Fzd−/− embryos; n = 2 embryos). Solid bars: control; open bars: Fzd−/− embryos.

Figure 6. Patterning of the ventral and distal optic vesicle occurs normally in Fzd5−/−embryos

At E10.5, Pax6 is expressed in the developing neural retina, lens (arrowhead in B) and presumptive RPE of control (A) and Fzd5−/− embryos (B). Similarly, Chx10 is present in the neural retina of control (C) and in the distal region of the optic vesicle in mutant embryos (D). Pax2 protein is present in the optic vesicle and stalk at 27 somites in mutant embryos (E) similar to the expression observed in control embryos (F). Ventral expression of Vax2 is also normal in Fzd5−/− optic vesicles at 30 somites (compare G, H).

Figure 7. Effects of germline and conditional inactivation of Fzd5 on FGF15, FoxE3 and Mab21L1 expression in the mouse optic vesicle

Whole mount in situ hybridization for FGF15 (A, B: 33 somites, C, D: 35 somites) and FoxE3 (E, F: 33 somites, G, H: 30 somites) showing lateral views. Coronal sections of FoxE3 (I, J: 30 somites) and Mab21L1 expression (K, L: 30 somites) are shown. Controls (A, C, E, G, I, K), embryos with a germline mutation (B, F, J, L) or a conditional disruption of Fzd5 (D, H) are presented. Germline disruption of Fzd5 leads to an absence of FGF15 (arrow in B), FoxE3 (arrow in F, arrowhead in J) and Mab21L1 expression (L, arrow) in the optic vesicle or lens placode. Embryos with conditional disruption of Fzd5 were generated using Six3-Cre heterozygous for the Fzd5 null allele and one floxed Fzd5 allele. Conditional Fzd5−/− embryos exhibit a normal expression pattern of FGF15 and FoxE3 in the optic vesicle (arrows in D, H, respectively).