Liu JT et al. (2011), Over-expression of atf4 in Xenopus embryos inte...

XB-ART-44146

Dongwuxue Yanjiu 2011 Oct 01;325:485-91. doi: 10.3724/SP.J.1141.2011.05485.

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Over-expression of atf4 in Xenopus embryos interferes with neurogenesis and eye formation.

Liu JT , Yang Y , Guo XG , Chen M , Ding HZ , Chen YL , Wang MR .

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Accumulated evidence indicates that the activating transcription factor 4 (atf4) is a developmentally relevant gene. Here, we report on the characterization of atf4 in Xenopus embryos, which is differentially expressed in the central nervous system, eyes, blood, and the pronephros, as well as in developing endodermal organs such as the stomach, duodenum, liver, and pancreas. Ectopic expression of atf4 in the animal hemisphere of Xenopus embryos had no obvious effects on the induction of neural progenitors, but suppressed neurogenesis and eye formation without promoting apoptosis. Our data suggest that tightly controlled atf4 activities may be crucial for normal neurogenesis and early eye patterning.

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Species referenced: Xenopus laevis
Genes referenced: atf4 irx1 otx2 pax6 rax six3 sox3 tubb2b
GO keywords: eye development [+]

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	Fig. 1 Spatial and temporal expression of atf4 in Xenopus embryos (A-L) Whole mount in situ hybridization analysis data. (A-E) Anterior views. (F, G) Lateral views. (H) Anterior view of an embryo transversally dissected at the position indicated by the white dashed line in G. (I) Lateral view. (J) Ventral view of the embryo in I showing of the expression in the blood island. (K) Isolated gut. (L) Ventral view. ba, branchial arches; bi, blood island; cg, cement gland; ey, eye; hb, hindbrain; li, liver; mb, midbrain; opv, optic vesicle; ov, otic vesicle; pa, pancreas; pd, pronephric duct; sc, spinal cord; sd, part of stomach and duodenum. (M) RT-PCR analysis reveals the temporal expression profile of atf4 during Xenopus embryogenesis. UE, unfertilized egg. NC, negative control. Ornithine decarboxylase (ODC) was used as the loading control.
	Fig. 2 Overexpression of atf4 interferes with neurogenesis and eye anlage formation (A-C) Lateral view of the embryos showing dose-dependent effects of atf4 on eye development. (D-N) Effects of atf4GR on the expression of various neural marker genes. The injected sides are on the right. Dexamethasone was added at stage 12.5. (D, F, H, J, L−N) Anterior views. (E, G, I, K) Dorsal views of D, F, H, and J, respectively. The white triangles in D, F, and H highlight the missing of marker gene expression in the lens placode, trigeminal ganglion, and placode on the injected side, respectively. The red dashed lines in L, M, and N illustrate the midline of the embryos. Dex, dexamethasone. The statistics for the phenotype are as follows: sox3 (19/20), N-tubulin (20/24), pax2 (32/34), pax6 (20/20), otx2 (18/21), six3 (16/18), rx1 (12/14).
	Fig. 3 Overexpression of atf4 led to microphthalmia (A−F) Lateral view. (A, B) The expression of sox3 in the lens is lost on the injected side labeled by red triangle. (C, D) The expression of pax6 in the retina is reduced on the injected side. (E, F) The retinal pigment epithelium is poorly developed on the injected side. uninj, uninjected side; inj, injected side.
	Fig. 4 Overexpression of atf4 in Xenopus embryos did not cause apoptosis At stage 13.5, the TUNEL signals detected are very weak in control embryos, as well as in atf4GR injected embryos with or without dexamethasone treatment. (A−F) Representative embryos from each group with the strongest TUNEL signals. B, D, and F are higher magnification views of A, C, and E, respectively. Statistics are indicated in B, D, and F for each group. All the rest embryos analyzed in each group showed very weak signals (data not shown). Before TUNEL assay, the embryos were subjected to betagalactosidase staining to trace the injected sites.
	atf4 (activating transcription factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior right, dorsal up.
	atf4 (activating transcription factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior right, dorsal up.