Schuff M et al. (2010), FoxO genes are dispensable during gastrulation ...

XB-ART-40616

Dev Biol 2010 Jan 15;3372:259-73. doi: 10.1016/j.ydbio.2009.10.036.

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FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis.

Schuff M , Siegel D , Bardine N , Oswald F , Donow C , Knöchel W .

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Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis.

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Species referenced: Xenopus laevis
Genes referenced: akt1 ap2a2 chrd foxd3 foxi1 foxo1 foxo3 foxo4 foxo6 gata5 myh6 myocd nkx2-5 otx2 pax6 sod1 sox17a sox2 tbx20 tbxt
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	Fig. 2. Expression of FoxO genes. (A) Temporal expression patterns of FoxO1, FoxO3, FoxO4 and FoxO6 genes during Xenopus embryogenesis and relative expression in adult tissues. Amount of transcripts was analyzed by semi-quantitative RT-PCR using RNA isolated from embryos at indicated developmental stages (Nieuwkoop and Faber, 1967) or from adult tissues. Expression levels are shown in relative units. Histone H4 was used as internal control. (B-M) Spatial patterns analyzed by whole mount in situ hybridization of FoxO4 (B-H) and FoxO6 (I-M). (B) 2-cell stage, lateral view. (C) Stage 17, anterior view. (D) Stage 21, anterior view. (E) Stage 23 embryo hybridized with a FoxO4 sense probe, anterior view. (F) Magnification of the head of an embryo, stage 29. (G) Stage 35/36, lateral view. (H) Transversal section of the head, stage 38. (I) Stage 22, anterior view. (J) Stage 27, lateral view. (K) Magnification of the head at stage 28, anterior view. (L) Stage 32, lateral view. (M) Stage 30, anterior view. Red arrows indicate the eye field, yellow arrows indicate the olfactory pits. ba, branchial arches; hg, hatching gland; hm, head mesenchyme; l, lens; nt, neural tube; ov, otic vesicle; pd, pronephric duct; pl, presumptive lens; pr, presumptive retina; r, retina.
	Fig. 3. Overexpression of FoxO1, FoxO4, FoxO6 and daf-16 interferes with early Xenopus development. (A) Whole mount in situ hybridization of uninjected control embryos and FoxO RNA injected embryos for Xbra, Sox17α, Sox2 at stage 11 (vegetal view), Xema, stage 11 (animal view), and Chordin at stage 15 (dorsal view). RNAs were bilaterally injected at 2-cell stage with 500 pg/blastomere (FoxO1), 400 pg/blastomere (FoxO4), 200 pg/blastomere (FoxO6) and 400 pg/blastomere (daf-16). The right column shows the phenotypes of unilaterally FoxO RNA injected embryos at stage 18, dorsal view. (B) Real time RT-PCR in gain of function embryos. Injected embryos were collected at stage 11.5. RT-PCR was performed for indicated genes. Percentages of phenotypes are given in supplementary Table S2.
	Fig. 4. FoxO overexpression phenotype depends upon Akt phosphorylation status. (A) Uninjected control embryo at stage 13. (B) Bilateral injection of 500 pg FoxO3 RNA does not reveal major phenotypic alterations. (C, D) Bilateral injection of 250 pg (C) or 500 pg FoxO3-TM RNA (D) prevents blastopore closure. (E) Uninjected control embryo at stage 11.5. (F) Bilateral injection of 400 pg FoxO4 prevents blastopore formation. (G) Bilateral injection of 300 pg IGF-1. (H) Co-injection 300 pg IGF-1 and 400 pg FoxO4 RNA restores blastopore formation. (I) Bilateral injection of 400 pg IGF-2. (J) Embryo, co-injected with 400 pg FoxO4 and 400 pg IGF-2 RNA, is rescued to wild type. (K) Bilateral injection of 500 pg Akt-1 RNA. (L) Embryo, co-injected with 400 pg FoxO4 and 500 pg Akt-1 RNA, is rescued to wild type. (M) Embryo, co-injected with 400 pg FoxO3-TM and 500 pg Akt-1 RNA, stage 11.5. The wild type phenotype cannot be restored. (N) Bilateral injection of 200 pg FoxO6 prevents blastopore formation. (O) Embryo, co-injected with 200 pg FoxO6 and 500 pg Akt-1. Blastopore formation fails. (P) Uninjected control embryo, stage 19. (Q) Bilateral injection of 500 pg Akt-1 RNA. (R) Embryo, co-injected with 400 pg FoxO4 and 500 pg Akt-1 RNA. Akt-1 prevents formation of the FoxO4 phenotype and allows neural plate formation. All bilateral injections were performed at 2-cell stage. (S) HeLa cells transfected with GFP, FoxO3-GFP, FoxO3-TM-GFP and FoxO6-GFP fusion-constructs. Percentages of blastopore closure are given in supplementary Table S3.
	Fig. 5. Loss of FoxO function results in severe developmental defects. (A) Left column: Uninjected control embryo at stage 39, bilaterally CoMO injected embryo (50 ng/blastomere), mm-FoxO4MO injected embryo (40 ng/blastomere). Right column: FoxO1MO injected embryo (50 ng/blastomere), FoxO6MO injected embryo (30 ng/blastomere), FoxO4MO injected embryo (20 ng/blastomere). All embryos are shown by lateral view. (B) Real time RT-PCR for Sod-2 in wild type and FoxO depleted embryos at stages 18 and 24. FoxO1MO: 35 ng/blastomere, FoxO4MO: 15 ng/blastomere, FoxO6MO: 25 ng/blastomere. (C) FoxO3 depleted embryo and rescue (anterior view). Uninjected control embryo and bilaterally CoMO injected embryo (50 ng/blastomere) at stage 19. Injection of 20 ng FoxO3MO does not interfere with blastopore closure but leads to death during neurulation. Co-injection of 20 ng FoxO3MO together with 500 pg FoxO3 RNA of X. tropicalis can rescue to wild type. (D) Rescue of eye structures in FoxO4 depleted embryos. Magnification of the head at stage 35/36 (lateral view): uninjected control; CoMO injected embryo; FoxO4MO injected embryo, the eye is absent; rescue of the eye by co-injection of 160 pg mutated FoxO4-GR RNA. 15 ng CoMO or FoxO4MO were co-injected with fluorescein in one dorsal blastomere at 4-cell stage. Percentages of phenotypes are given in supplementary Table S4-S6.
	Fig. 6. FoxO4MO injection affects eye, heart and cranial crest development. (A) FoxO4 depletion results in lack of eye structures. Head of an unilaterally FoxO4MO injected embryo (40 ng/blastomere) at stage 39 (lateral view). The eye is almost absent. Middle: Pax-6 whole mount in situ hybridization of control and unilaterally FoxO4MO injected embryos at stage 21. Bottom: Otx-2 whole mount in situ hybridization of control and unilaterally FoxO4MO injected embryos at stage 25. (B) FoxO4 depletion affects cranial crest and jaw development. In situ hybridization of unilaterally FoxO4MO injected embryos with the neural crest markers FoxD3 at stage 23 and AP2α at stage 33. Red arrows indicate the lack of FoxD3 and AP2α expression. Bottom: Alcian blue stained control and unilaterally FoxO4MO injected embryo (30 ng/blastomere) at stage 47. (C) FoxO4 depletion results in failure of heart development. Top: lateral view on control and bilaterally FoxO4MO injected embryo at stage 41. Right: control heart of stage 43 embryo and heart of FoxO4MO injected embryo (20 ng/blastomere at 2-cell stage). Bottom: control- and unilaterally FoxO4Mo injected embryos. myocd and MHCα in situ hybridization reveals reduced expression at the injected side. In situ hybridization of unilaterally FoxO4MO injected embryos with Nkx2.5, GATA-5 and Tbx20. Red asterisks indicate the side of injection. ba, branchial arches; c, conus; ch, ceratohyale; mc, Meckel's cartilage; v, ventricle. Percentages of phenotypes are given in supplementary Table S7.
	Fig. 7. BrdU incorporation demonstrates reduced cell proliferation following FoxO overexpression and depletion. (A) Animal view of control embryo, stage 9.5. GOF embryos were bilaterally injected at 2-cell stage with 500 pg/blastomere FoxO1 or FoxO3, 250 pg/blastomere FoxO3-TM, 400 pg/blastomere FoxO4 and 200 pg/blastomere FoxO6 RNA. (B) Bilaterally CoMO and FoxO3MO injected embryos at stage 11.5 (animal view) and stage 15 (dorsal view). (C) CoMO, FoxO1MO and FoxO6MO injected embryo at stage 28 (lateral view). (D) Unilaterally FoxO4MO injected embryo at stage 27. (E) Magnification of the head of an unilaterally FoxO4MO injected embryo at stage 30 (lateral view). Morpholinos were uni- or bilaterally injected at indicated amounts into 2-cell stage embryos. Note that the blue spots represent BrdU positive cells, while the brown color is due to natural pigmentation of embryos. Asterisks indicate the morpholino injected side. ba, branchial arches; e, eye; n, neural fold.
	Fig. 8. FoxO proteins affect apoptosis. (A) Embryos were injected at 2-cell stage with the same amounts as given in Fig. 7. At stage 11.5, embryos were analyzed for apoptosis by TUNEL (animal view) and for caspase activities using caspase 8, 9 and 3/7 Glo assays. (B) Embryos injected with FoxO3MO and corresponding controls were analyzed by TUNEL at stage 15 (dorsal view, left), embryos injected with FoxO1MO, FoxO4MO or FoxO6MO were analyzed at stage 26 (lateral view, bottom). Caspase 8, 9 and 3/7 activities were determined at stages 19 and 26, respectively.
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