Yokota C et al. (2017), Apolipoprotein C-I mediates Wnt/Ctnnb1 signalin...

XB-ART-53828

Int J Dev Biol 2017 Jan 01;616-7:415-425. doi: 10.1387/ijdb.160399cy.

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Apolipoprotein C-I mediates Wnt/Ctnnb1 signaling during neural border formation and is required for neural crest development.

Yokota C , Åstrand C , Takahashi S , Hagey DW , Stenman JM .

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In vertebrates, the neural crest and placodes originate in the neural border, which is located between the neural plate and epidermal ectoderm. The neural crest and placodes give rise to a vast array of cell types. Formation of neural crest is a multi-step process, in which Wnt signals are used reiteratively, but it is currently not clear if a Wnt signal is required for neural border formation. Here, we have identified apolipoprotein C-I (apoc1) in a screen for genes regulated by Wnt/Ctnnb1 signaling in late blastula stage Xenopus tropicalis embryos. We show that Xenopus laevis apoc1 encodes a small, secreted protein, and is induced by Wnt/Ctnnb1 signaling. Depletion of Apoc1 protein results in a neural border formation defect and loss of border fates, including neural crest cells. However, unlike another Wnt/Ctnnb1 target, gbx2.2, apoc1 is not required for patterning of the neural border. We further show that gbx2.2 and apoc1 are independently regulated by Wnt signaling. Our results thus suggest that Wnt regulates border formation and patterning by distinct genetic mechanisms.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: apoc1 axin1 chrd.2 ctnnb1 dkk1 foxd3 foxi4 foxi4.2 gbx2.2 id3 krt12.4 lrp6 msx1 myc nodal3.2 nodal3.4 odc1 pax3 snai1 snai2 sox2 sox9 twist1 wnt1 wnt3a zic1
GO keywords: ectodermal cell fate determination [+]
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	Fig. 1. Identification, characterization and regulation of Xenopus apoc1. (A-C) Bar charts showing the expression levels, displayed as fragments per kilobase of transcript per million fragments mapped (FPKM), for apoc1 (A), the Wnt regulated gene nodal3.2 (B), and the non-Wnt regulated gene odc1 (C) in Xenopus tropicalis embryos that were either uninjected (blue), injected with 105 pg axin1 mRNA (pink) or 24 pg <N-HA-ctnnb1 mRNA (green). (D) Schematic drawing of the Apoc1 secretion experiment. mRNA was injected at the two-cell stage. The blastopore fluid was collected at the gastrula stage and subjected to western blot (WB) analysis to determine whether flag-tagged Apoc1 protein is secreted from cells during embryonic development. (E) Apoc1, but not Ctnnb1, protein was detected by WB in the blastocoel fluid after injection of flag-tagged apoc1 (500 pg) and flag-tagged ctnnb1 (1 ng). The tubulin blot shows that there is no cellular contamination in the blastopore fluid. (F) Wnt regulation of apoc1 in Xenopus laevis embryos. (F) 300 pg of wnt3a or 200 pg of chrd and 300 pg wnt3a were injected into the animal side of two blastomeres at the two-cell stage. Animal cap explants were dissected from stage 9 embryos, cultured until stage 10, and processed for RT-PCR analysis. Expression of apoc1, and the known Wnt target gene nodal3.2, was induced by wnt3a alone, and chrd+wnt3a. Odc1 was used as loading control. (G) Apoc1, gbx2.2 and pax3 are immediate-early target gene of Wnt/Ctnnb1 signaling. 200 pg of GR-ctnnb1 and 100 pg lacZ mRNA were injected into one side of a dorsoanimal blastomere of 8-cell stage embryos and the embryos were treated with cycloheximide 30 min prior to DEX treatment at stage 11.5. The expression of apoc1, gbx2.2, pax3 and snail2 were examined by whole mount in situ hybridization. Orange arrows indicate ectopic localization of the tested genes.
	Fig. 2. Spatiotemporal expression pattern of Xenopus laevis apoc1. (A) The temporal expression pattern of apoc1 during Xenopus development (4-cell stage, stages 10-32) was determined by RT-PCR (RT- is without reverse transcriptase from stage 32). (B-P) The spatiotemporal expression pattern of apoc1 determined by whole mount in situ hybridization. (B,C) Apoc1 expression at stage 11; bp - the closing blastopore. Anterior view (D-G), dorsal view (H-K), posterior view (L-O) of stage 14 (D,H,L), stage 15 (E,I,M), stage 17 (F,J,N) and stage 20 (G,K,O) embryos. (P) Lateral view of a stage 30 embryo.
	Fig. 3. Loss of neural crest derivatives in Apoc1 depleted embryos. (A) The Apoc1 translational blocking MO (tbMO) targets the 5’ UTR region of the Xenopus laevis apoc1 gene. To determine the efficiency of the tbMO, two flag-tagged apoc1 constructs were used differing only in the region upstream of the ATG start site. (B) Western blot using an anti-flag antibody on lysates from embryos injected with 300 pg 5’UTR-apoc1-flag or apoc1-flag mRNA and 40 ng Apoc1 tbMO or 40 ng control MO (ctrl). (C) 30 ng of the splice blocking morpholino (spMO) or control MO (ctrl) was injected into the animal side of two blastomeres at the two-cell stage and the embryos were assayed by RT-PCR at stage 10.5. Odc is used as loading control. (D) Schematic drawing of the experiment. 40 ng Apoc1 MO was injected into either the presumptive head (F,G) or trunk/tail (H,I) region together with 200 pg lacZ mRNA. (E) Embryos injected with 40 ng control MO were normal. (F,G) Injections targeted to the head region resulted in 76.1% (n=134) of the embryos displaying small or missing eyes and head deformation. (H,I) Injections targeted to the trunk region resulted in perturbed dorsal fin development in 55.2% (n=38) of the embryos. The pictures in (G,I) were taken prior to X-gal staining (F,H).
	Fig. 4. Apoc1 is required for neural border formation and neural crest emergence. (A) Whole mount in situ hybridization for the neural crest (NC) markers c-myc, sox9, snai2, twist1, id3. Control MO injected embryos are shown in upper panel and Apoc1 tbMO injected ones are shown in lower panel. (B) The reduction of sox9 and snai2 expression by the Apoc1 tbMO could be rescued by coinjection of 400 pg of apoc1 mRNA (upper panel) but not by coinjection of 400 pg of sp-apoc1 mRNA (lower panel). (C) Whole mount in situ hybridization for the neural plate border specifiers msx1, pax3, zic1, the neural marker sox2, and the non-neural ectoderm marker xk81a1. Control MO injected embryos are shown in upper panel and Apoc1 tbMO injected ones are shown in lower panel. One of the dorsal blastomeres was injected with 30 ng of either control MO (upper panels in A, C) or Apoc1 tbMO (lower panels in A, C) together with 100 pg lacZ mRNA. Embryos were fixed at stage 15. The injected side was traced by X-gal staining (blue).
	Fig. 5. Apoc1 can substitute for wnt in the animal cap neural crest induction assay. (A) 200 pg of chrd, 200 pg wnt3a and/or 300 pg of apoc1 was injected into two animal blastomeres of 4- or 8-cell stage embryos. Animal caps were dissected at stage 9 and cultured until stage 19. Gene expression was assayed by RT-PCR. (B) 125 pg tBR (dominant negative form of bmpr1a) and 30 pg aN-HA-Ctnnb1 (constitutively active form of ctnnb1) mRNA was injected in two animal blastomeres at the 4- to 8-cell stage to induce neural crest cell marker expression. In addition, 40 ng of either control (ctrl) or translation blocking Apoc1 MO (tbMO) with or without 400 pg MO-resistant apoc1 mRNA was injected to determine if Apoc1 is required for neural crest induction. Animal caps were dissected at stage 9 and cultured until stage 16. Gene expression was assayed by RT-PCR. (C) Coinjection of apoc1 and zic1 into the ventral side of the embryo induced ectopic expression of sox9 and snai2. 50 pg of zic1 +/- 500 pg of apoc1 was injected into ventral marginal cells of 8 cell-stage embryos. The embryos were fixed at stage 14, and subjected to whole mount in situ hybridization. The blue arrows point to endogenous sox9 and snai2 expression. The yellow arrowheads show the area of ectopic sox9 and snai2 expression.
	Fig. 6. Apoc1 and gbx2.2 are required for emergence of neural crest cells, but are independently regulated by Wnt signaling. (A-H) The expression of apoc1 and gbx2.2 is independently regulated, but both genes are required for neural crest induction. Apoc1 expression in Gbx2.2 (A) or control MO (B) injected embryos and gbx2.2 expression in Apoc1 (C) or control MO (D) injected embryos. The reduction of sox9 and snai2 expression was not rescued by coinjection of 100 pg gbx2.2 mRNA in Apoc1 (E,G) or 400 pg apoc1 mRNA in Gbx2.2 (F, H) depleted embryos. 30 ng of MO and 100 pg lacZ mRNA was injected into one side of a single dorsoanimal blastomere of 8-cell stage embryos. The embryos were fixed at stage 15. (I-R) Apoc1 and gbx2.2 are both required for Wnt-mediated neural crest induction in vivo. 200 pg of GR-ctnnb1 and 100 pg lacZ mRNA were injected into one side of a dorsoanimal blastomere of 8-cell stage embryos and the embryos were treated with DEX at stage 10.5. The embryos were fixed at stage 15 for whole mount in situ hybridization with snail2 (I), sox9 (J), apoc1 (K), gbx2.2 (L), msx1 (M), and pax3 (N). Ectopic expression of gbx2.2 in GR-ctnnb1 injected embryos was not reduced by coinjecting 30 ng Apoc1 tbMO (P), but the anterior expansion of snai2 was rescued (O). Ectopic expression of apoc1 in GR-ctnnb1 injected embryos was not reduced by coinjecting 30 ng Gbx2.2 MO (R), but the anterior expansion of snai2 was rescued (Q). Blue arrowhead indicates ectopic expression.
	Fig. 7. Apoc1 does not posteriorize the border. (A-C) Expression of apoc1 (400 pg) is not sufficient to posteriorize the anterior neural border as demonstrated by analysis of snai2 (A), pax3 (B) and foxi4.1 (C) using in situ hybridization. One of the dorsoanimal blastomeres was injected with 400 pg apoc1 and 100 pg lacZ mRNAs at the 8-cell stage. (D-F) Apoc1 is required for expression of foxi4.1 in the posterior preplacodal domain. Whole mount in situ hybrydization of foxi4.1 in control MO (D) or Apoc1 tbMO (E,F) injected embryos. The yellow arrow shows the posterior limit of foxi4.1 expression on the uninjected side. 30 ng of MO was injected with 100 pg of lacZ mRNA into one side of a dorsoanimal blastomere of 8-cell stage embryos and fixed at stage 15. (G) 200 pg of chrd, 200 pg wnt3a and/or 300 pg of apoc1 was injected into two animal blastomeres at the 4- to 8-cell stage. Animal caps were dissected at stage 9 and cultured until stage 14. Gene expression was assayed by RT-PCR. Odc1 is a loading control.
	Fig. S1. The apoc1 spMO morphants resulted in identical phenotypes as tbMO morphants. (A) Schematic drawing of the experiment. 40 ng Apoc1 spMO was injected into either the presumptive head (C) or trunk/tail (D). (B) Embryos injected with 40 ng control MO were normal. (C) Injections targeted to the head region resulted in 56.4% (n=55) of the embryos displaying small or missing eyes and head deformation. (D) Injections targeted to the trunk region resulted in perturbed dorsal fin development in 45.2% (n=31) of the embryos.
	Fig. S2. Apoc1 is required for neural border formation and neural crest emergence. Whole mount in situ hybridization for the neural crest (NC) markers snai2, sox9 and twist1 and the neural plate border specifiers pax3 and zic1. Control MO (40ng) injected embryos are shown in upper panel and Apoc1 spMO (40ng) injected ones are shown in lower panel. Injection of Apoc1 spMO also results in a reduction of the expression of the NC markers as well as the neural plate border specifiers on the injected side.
	Fig. S3. Apoc1 mutant lacking signal sequence cannot be secreted. (A) Schematic drawing of the Apoc1 secretion experiment. mRNA was injected at the two-cell stage. The blastopore fluid was collected at the gastrula stage and subjected to western blot (WB) analysis to determine whether mutant flag-tagged Apoc1 protein (lacking signal sequence) is secreted from cells during embryonic development. (B) Apoc1, but not mutant protein was detected by WB in the blastocoel fluid after injection of 500pg of flag-tagged apoc1 or flag-tagged mutant apoc1 mRNA. The tubulin blot shows that there is no cellular contamination in the blastopore fluid.
	Fig. S4. Apoc1 does not cooperates with extracellular Wnt components to modulates Wnt signaling in TOPFlash assay. (A) Schematic drawing of the experiment. (B) 300 pg of lrp6 mRNA and either 20 pg of wnt3a or 50 pg dkk1 with or without 500 pg of apoc1 mRNA were co-injected with 100 pg of 14XTOPFlash and 25 pg Renilla reporter DNA into 4 to 8cell-stage Xenuous embryos. Firefly and Renilla luciferase were measured at st.10.