Watanabe T et al. (2018), Fam46a regulates BMP-dependent pre-placodal ect...

XB-ART-55348

Development 2018 Oct 26;14520:. doi: 10.1242/dev.166710.

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Fam46a regulates BMP-dependent pre-placodal ectoderm differentiation in Xenopus.

Watanabe T , Yamamoto T , Tsukano K , Hirano S , Horikawa A , Michiue T .

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The pre-placodal ectoderm (PPE) is a specialized ectodermal region which gives rise to the sensory organs and other systems. The PPE is induced from the neural plate border during neurulation, but the molecular mechanism of PPE formation is not fully understood. In this study, we examined the role of a newly identified PPE gene, Fam46a, during embryogenesis. Fam46a contains a nucleoside triphosphate transferase domain, but its function in early development was previously unclear. We show that Fam46a is expressed in the PPE in Xenopus embryos, and Fam46a knockdown induces abnormalities in the eye formation and the body color. At the neurula stage, Fam46a upregulates the expression of PPE genes and inhibits neural crest formation. We also show that Fam46a physically interacts with Smad1/Smad4 and positively regulates BMP signaling. From these results, we conclude that Fam46a is required for PPE formation via the positive regulation of BMP signaling. Our study provides a new mechanism of ectodermal patterning via cell-autonomous regulation of BMP signaling in the PPE.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 bmpr2 chrd dnai1 eef1a1 eef1a2 eya1 foxd3 krt12.4 myc ncam1 npb odc1 pax2 pax3 pax6 pax8 rpe six1 six6 smad1 smad10 smad2 smad4 snai2 sox2 sox3 tent5a ventx1.2 ventx2.2 wnt8a zic1
GO keywords: camera-type eye development [+]
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	Fig. 1. Temporal and spatial expression pattern of Fam46a [tent5a] in Xenopus. (A) RT-PCR analysis of Fam46a expression. The expression of Fam46a is faintly detected from the maternal stage to stage 9, and increased from stage 10, similar to that of Zic1 (NPB), Vent1, Vent2 (BMP target gene), and Chordin (BMP antagonist). The expression of PPE marker genes, Six1 and Eya1, increased soon after the onset of Fam46a expression. ODC was used as a control. (B-E) Whole-mount in situ hybridization (WISH) was performed with a Fam46a (B, B’) and Six1 (C, C’) probe. The anterior view (B, C); the lateral view (D, E); the hemi-section (B’, C’, D’, D”, E’). Development • Accepted manuscript The stage of the embryo was shown in each panel. PPE, pre-placodal ectoderm; Op, optic vesicle; Ot, otic vesicle; Le, lens; Br, branchial arch mesenchyme; RPE, retinal pigment epithelium. (F) HeLa cells were transfected with GFP-Fam46a and cultured for 24 hours. Nuclei were stained with NUCLEAR-ID Red. Scale bars represent 20 μ m.
	Fig. 2. RT-qPCR analysis of Fam46a and other ectodermal marker genes in the PPE-like cells. 20 pg Chordin (Chd) mRNA was injected into all blastomeres of the four-cell stage embryos. The animal caps were dissected at stage 9 and cultured to stage 15 with/without 25 μM SU5402 (FGF inhibitor). The expression of Fam46a, PPE genes (Six1 and Eya1), an epidermis gene (XK81), neural plate genes (Sox2, NCAM), and NC genes (FoxD3, Snail) was assessed by RT-qPCR. EF1α was used as a control. Unpaired two-tailed t-tests were used to determine the statistical significance (P <0.05, P < 0.01, **P < 0.001) ( n=10, 3 biological replicates, Error bars represent S.E.).
	Fig. 3. The phenotypes of gain- and loss-of-function of Fam46a. (A-B) Overexpression of Fam46a caused head defects, small eyes, and abnormal pigmentation. Lateral views of tadpole embryos injected with 500 pg of Fam46a mRNA or LacZ mRNA into the animal-dorsal blastomeres at the four-cell stage. (C-D) Knockdown of Fam46a caused retinal defects and abnormal pigmentation. Lateral views of tadpole embryos injected with 20 ng of Fam46a MO or standard MO into the animal-dorsal blastomeres at the four-cell stage. (E) The ratio of the phenotypes of Fam46a mRNA-injected embryos were summarized as bar graphs. Severe phenotype includes absence of eyes, head expansion, and abnormal pigmentation. Weak phenotype means the embryos that showed only abnormal pigmentation. (F) The design of Fam46a MO. Upper diagram shows the binding position of Fam46a MO (red). Gray box indicates the exon (with the exon number) and black line indicates the intron, Development • Accepted manuscript whose length is 1.3 kb. This MO was designed to block the splicing of both Fam46a.L and Fam46a.S. The lower image shows the result of RT-PCR using Standard MO (Std)- or Fam46a MO (Fam)-injected embryos. (G) The ratio of the phenotypes of Fam46a MO-injected embryos is summarized as bar graphs. The embryos that showed retina defects and abnormal pigmentation were classified into “severe phenotype”. The specificity of MO effects was confirmed by rescue experiments, in which Fam46a MO was co-injected with Fam46a mRNA.
	Fig. 4. Fam46a is required for the PPE but inhibits the NC specification. (A-C) WISH analysis of Six1 (PPE), Slug (NC), FoxD3 (NC), XK81 (Epidermis), and Sox3 (Neural plate) expression patterns in Xenopus embryos injected with Fam46a mRNA or Fam46a MO (with a tracer (LacZ)). Embryos were injected at the four-cell stage and collected at the early neurula stage (Stage 15). The asterisk denotes the injected side, which was labeled with β-galactosidase (β-gal) staining. The sample sizes are as follows: Six1 (Fam46a n=58/102, Fam46a MO n=81/131), Slug (Fam46a n=25/60, Fam46a MO n=21/55), FoxD3 (Fam46a n=24/59, Fam46a MO n=21/37), XK81 (Fam46a n=9/9, Fam46a MO n=9/24), Sox3 (Fam46a n=7/ 22, Fam46a MO n=10/18). (D) Schematic figures of the ectodermal pattern in Fam46a mRNA or Fam46a MO-injected embryos, referred to A-C. The asterisks denote the injected side. (A, anterior; P, posterior; red, PPE; green, NC; gray, Neural plate; white, Epidermis.) (E) Embryos at the four-cell stage were injected in their animal Development • Accepted manuscript side with Chordin (Chd) and Fam46a MO. The animal caps were dissected at the blastula stage and were cultured until early neurula stage (Stage15) with/without SU5402.Then the expression level of Zic1 (NPB), Six1 (PPE), Six6 (anterior PPE), and Pax2 (posterior PPE) were analyzed by RT-qPCR (n=20, 3 biological replicates, error bars represent S.E.). Each value was normalized to the level of EF1α expression. (F) Chd, Wnt8, and Fam46a mRNA were injected into the animal side of the four-cell stage embryos. The animal caps were dissected at the blastula stage and the specimens were cultured as in E. Then the expression level of NPB genes (Pax3 and Zic1) and NC genes (Snail and Slug) were analyzed by RT-qPCR (n=20, 3 biological replicates, error bars represent S.E.). Each value was normalized to the level of EF1α expression. Unpaired two-tailed t-tests were used to determine the statistical significance (P <0.05, P < 0.01, P < 0.001, **p<0.0001).
	Fig. 5. Fam46a regulates PPE specification via modulating BMP signaling. (A, B) The expression of Vent1 and Vent2 at blastula stage. Embryos were injected with 500 pg of BMPRII mRNA, tBR mRNA, Fam46a mRNA, or 5 ng of Fam46a MO (with a tracer (LacZ)) at the four-cell stage. The specimens were collected at stage 10.25. Dashed circles indicate the injected cells, colored with Red-gal. All images are indicated in the animal-pole view (the upper side is the dorsal side, and vice versa). Arrowhead indicates the ectopic expression of Vent1 in BMPRII-expressed embryos. Endogenous expression of Vent2 was also observed in the ventral region (U-shape). Vent1 (BMPRII n=8/9, tBR n=11/11, Fam46a n=14/16, Fam46a MO n=19/19), Vent2 (BMPRII n=17/19, tBR n=12/13, Fam46a n=13/17, Fam46a MO n=16/16). (C) RTqPCR analysis of BMP-signal activity using the animal cap that was injected with BMP4 and Fam46a mRNA. The explants were collected at the early neurula stage (Stage 15) and analyzed by RT-qPCR (n=20, 3 biological replicates, error bars represent S.E.). Each value was normalized to the level of EF1α expression. Unpaired two-tailed t-tests were used to determine statistical significance. (P <0.05, P < 0.01, **P < 0.001) (D) WISH experiments examining Six1, FoxD3, XK81, and Sox2 expression patterns in the embryos injected with 250 pg of tBR mRNA or 250 pg tBR mRNA + 250 pg of Fam46a mRNA. Embryos were injected at the four-cell stage and were collected at the early neurula stage (Stage 15). An asterisk denotes the injected side with β-galactosidase (β-gal) staining as the lineage tracer. Six1 (tBR n=8/10, tBR + Fam46a n=6/10), FoxD3 (tBR n=5/11, tBR + Fam46a n=10/12), XK81 (tBR n=10/10, tBR + Fam46a n=10/10), Sox2 (tBR n=9/ 9, tBR + Fam46a n=8/9). (E) Ectoderm pattern diagram in tBR mRNA or tBR mRNA+ Fam46a mRNAinjected embryos, reffered to D. Asterisk denotes the injected side.
	Fig. 6. Fam46a interacts with Smad1 and Smad4 in Xenopus embryos. (A, C) Immunoprecipitation assays were performed using the lysates of the embryos injected with GFP, GFP-Fam46a or GFP-Smad4, and Myc-Smad1 mRNA, which had been cultured until the early gastrula stage (Stage 10.5) (n=10) with/without 100 nM LDN-193189. (B) Quantitative analysis of the precipitates of GFP or GFP tagged proteins in A by ImageJ (3 biological replicates, error bars represent S.E.). Each value was normalized to the level of each input protein. (D) Schematic figure of the construct of Myc-Smad1-WT, Myc-Smad1-MH1, and Myc-Smad1-MH2. (E) An immunoprecipitation assay using the embryos injected with Myc, Myc-Smad1-WT, Myc-Smad1-MH1, Myc-Smad1-MH2, and GFP-Fam46a mRNA and cultured until early gastrula stage (Stage 10.5) (n=10). (F) An immunoprecipitation assay using the embryos injected with GFP, GFP-Fam46a, Smad2-GFP, and Myc-Smad4 mRNA and cultured until Stage 10.5 (n=10). The asterisk indicates a nonspecific band. (G) Quantitative analysis of the precipitates of GFP or GFP tagged proteins in F by ImageJ (3 biological replicates, error bars represent S.E.). Each value was normalized to the level of each input protein.
	Fig. 7. Fam46a regulates Smad1 stabilization. (A, B) Luciferase assay using the Vent2-TCFm-Luc reporter construct in the presence of Chordin, BMP4, Fam46a, or Fam46a MO. Embryos were injected at four-cell stage and were incubated to early gastrula stage (Stage 10.5) (n=10, 3 biological replicates, error bars represent S.E.). Unpaired two-tailed t-tests were used to determine statistical significance (P < 0.05, P < 0.01). (C, D) Western blot analysis using embryos injected with Myc-Smad1(C) or Myc-Smad4 (D) and GFPFam46a (C, D) at the four-cell stage and cultured until early gastrula stage (Stage 10.5) to examine the amount of Myc-Smad1 or Myc-Smad4 (n=10 each). β-tubulin was used as a control. The asterisks indicate phosphorylated Smad. (E) Quantification of phosphorylated and unphosphorylated Smad1 in C. Each value was normalized to the level of β-tubulin expression (3 biological replicates, error bars represent S.E.). Unpaired two-tailed t-tests were used to determine statistical significance (P <0.05). (F) A schematic model of BMP signaling regulated by Fam46a.
	Fig. S1. Alignment of amino acid sequences of Fam46 proteins. (A, B) Alignment of the homeologues, Fam46a.L and Fam46a.S using ClustalW. The identity of the amino acid sequences of the NTP transferase-7 domain and the Cterminal region of xFam46a.L and xFam46a.S was indicated (A). The alignment of the cDNAs of the homeologues (B). (C, D) Alignment of the orthologs of Fam46a. Xenopus (x); human (h); mouse (m); Drosophila (d; dMel-C). The identity of the amino acid sequence in the NTP transferase-7 domain and C-terminal region was indicated (C). The alignment of the cDNAs of the orthologues (D). (E, F) Alignment of the paralogues of Fam46 proteins. The identity of the amino acid sequences of the NTP transferase-7 domain and the C-terminal region was indicated (E). Identical and similar amino acid residues are marked with asterisk and double dots, respectively (F). Gaps are indicated by dashes (F). Amino acids of yellow background are predicted phosphorylation sites (F).
	Fig. S2. Whole-mount in situ hybridization (WISH) using Fam46a sense probe. (A-C) WISH was performed with a Fam46a sense probe. Anterior view (A); Lateral view (B, C). The embryonic stages are shown in each panel. The siblings stained with the anti-sense probe were shown in Fig.1.
	Fig.S3. The statistical analysis of Fam46a overexpressing embryos. (A, B) Examples of the measurement of the eye area (A) and the body length (B) in Fig. 3. Arrowheads and red regions denote the eye area. White bars indicate body length. These were quantified by ImageJ (C). Unpaired two-tailed t-tests were used to determine the statistical significance (n=3) (P <0.05, *P < 0.01).
	Fig. S4. Fam46a upregulates each PPE marker gene expression and inhibits other ectodermal gene expression. (A, B) Whole-mount in situ hybridization experiments examining the expressions of Six3 (anterior PPE) (n=34/79), Ath-3 (lateral PPE) (n=19/22), Pax8 (posterior PPE) (n=18/22), Pax3 (NPB) (n=12/34), Pax6 (eye-field) (n=11/21), and CG (cement gland) (n=21/67) in Xenopus embryos injected with Fam46a MO. Embryos were injected at the four-cell stage and were collected at early neurula stage (Stage 15). The asterisk denotes the injected side with β-galactosidase (β-gal) staining as the lineage tracer.
	Fig. S5. Fam46a is important for PPE marker gene expression and inhibits NC gene expression. (A) In situ hybridization in the PPE-like cells examining the expressions of the PPE genes (Six1 and Eya1). Embryos were injected with/without Fam46a MO at the four-cell stage. Then animal caps were dissected at the blastula stage and cultured until early neurula stage (Stage 15, n=10). Consistent with the results of RT-qPCR (Fig. 2), the PPE genes were highly expressed in the PPE-like cells, but the knockdown of Fam46a reduced the expressions. (B) In situ hybridization in the NC-like cells examining the expressions of the NC genes (Snail and Slug). Embryos were injected with/without Fam46a mRNA at the four-cell stage. Then animal caps were dissected at the blastula stage and cultured until early neurula stage (Stage 15, n=10). The NC genes were highly expressed in the NC-like cells, but the overexpression of Fam46a reduced the expressions.
	Fig. S6. Fam46a functions in the downstream of BMP signaling. (A, B) The Fluorescence and DIC images of HeLa cells. The cells were transfected with EGFP-Fam46a. At 24 h after transfection, the cells were treated with/without BMP4. The nuclei were visualized with NUCLEAR-ID Red. Images were captured before adding BMP4 (100 ng/ mL), 0 min, 60 min, and 120 min after adding BMP4. Scale bars, 10 μm. (C) The quantification of EGFP-Fam46a nuclear translocation. The nuclear/cytoplasm EGFP-Fam46a fluorescence intensity ratios were calculated and compared between pre- and 120 min-images. Unpaired two-tailed t-tests were used to determine the statistical significance. (P < 0.01). All error bars represent S.E. of the mean. (D) The animal cap assay examining Fam46a expression with the injection of BMP4 mRNA. The explants were cultured to early neurula stage (Stage 15) and the expression level of Fam46a were quantified by RT-qPCR (n=20, 3 biological replicates, error bars represent S.E.). Each value was normalized by EF1α expression level. Unpaired two-tailed t-tests were used to determine the statistical significance. (P < 0.01)
	Fig. S7. Fam46a expression promotes Smad1 nuclear translocation. (A) β-catenin MO, mCherry-Smad1 and NLS-iRFP mRNA were injected at four-cell stage. These embryos were co-injected with/without Chd or Chd + Fam46a mRNA. They were cultured until early gastrula stage (Stage 10.5) and observed using a confocal microscope. (B) The density ratio (nucleus vs cytoplasm) of mCherrySmad1 was quantified using ImageJ (3 biological replicates, error bars represent S.E.). Unpaired two-tailed t-tests were used to determine the statistical significance. (***P < 0.001.)
	tent5a (terminal nucleotidyltransferase 5A) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 28, anterior left.
	tent5a (terminal nucleotidyltransferase 5A) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 15, anterior view, dorsal up.
	tent5a (terminal nucleotidyltransferase 5A) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 28, transverse section, dorsal up.
	tent5a (terminal nucleotidyltransferase 5A) gene expression in Xenopus laevis, assayed by in situ hybridization, NF stage 28, transverse section, dorsal up.