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. 2020 Dec 9;10:21555. doi: 10.1038/s41598-020-78019-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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TFG regulates antero-posterior (A-P) patterning in the Xenopus embryo and is required for Wnt activity. (A) Diagram showing the TFG morpholino (MO) target sequence (in red), which is the same for both Xenopus L and S homeologs and span the ATG initiation codon. (B) Stage 24 embryos injected with control morpholino (Co-MO), showing normal development (92%, n = 127). (C) Embryos injected with 36 ng of TFG-MO showed severe enlargement of the head region (65%, n = 196). (D) Normal antero-posterior development was rescued by co-injecting 400 pg/embryo of human Flag-tagged TFG mRNA (77%, n = 52). (E) Embryos injected with 50 pg of mRNA encoding the Wnt antagonist Dkk1 showed head enlargement (100%, n = 35). (F,G) Embryos injected unilaterally with 18 ng of Co-MO or TFG-MO, together with 200 pg of nuclear LacZ mRNA, processed for in situ hybridization for Engrailed-2 (En2) and Krox20. LacZ lineage tracing (in red) shows the injected side. TFG-MO reduces En2 expression and shifts Krox20 posteriorly in 65% of the embryos (n = 20), compared to control MO (n = 33). (H,I) In situ hybridization for the pan-neural marker Sox2 at stage 20 showing enlargement of the neural plate following injection 4 times at the 2-cell stage with a total of 72 ng of TFG-MO per embryo (70%, n = 65), compared to the Ctrl-MO embryos (n = 35). (J) Co-injection with 800 pg of hTFG mRNA rescued the neural plate expansion (78%, n = 23). (K,M) Control embryos at stage 22 showed normal eye (Rx2a) and somite (MyoD) development (n = 18), while embryos injected with 32 pg of xWnt8 DNA together with 72 ng of scrambled Ctrl-MO showed reduced or no Rx2a expression, but maintained MyoD expression (66%, n = 18). However, embryos injected with the same amount of Wnt8 DNA together with 72 ng of TFG-MO showed rescue of Rx2a expression (84%, n = 32), indicating that TFG is required for xWnt8 signaling. (N–P) Embryos injected with Bighead morpholino (BH-MO) showed reduced head and cement gland development (71%, n = 21), compared to control embryos (n = 37), due to an increase in endogenous Wnt signaling (31). Co-injecting TFG-MO rescued normal head development in Bighead MO embryos (90%). Scale bars represent 500 µm.