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Pre-placodal ectoderm (PPE), a horseshoe-shaped narrow region formed during early vertebrate development, gives rise to multiple types of sensory organs and ganglia. For PPE induction, a certain level of FGF signal activation is required. However, it is difficult to reproducibly induce the narrow region with variations in gene expression, including FGF, among individuals. An intracellular regulatory factor of FGF signaling, Dusp6, is expressed by FGF signal activation and inactivates a downstream regulator, ERK1/2, in adult tissues; however, its role in early development is not well known. Here, we reveal that Dusp6 is expressed in an FGF-dependent manner in Xenopus PPE. Gain- and loss-of-function experiments showed that Dusp6 is required for expression of a PPE gene, Six1, and patterning of adjacent regions, neural plate, and neural crest. To reveal the importance of Dusp6 in variable FGF production, we performed Dusp6 knockdown with FGF-bead implantation, which resulted in varying Six1 expression patterns. Taken together, these results suggest that Dusp6 is required for PPE formation and that it contributes to the robust patterning of PPE by mediating FGF signaling.
Fig. 1. FGF-dependent expression of Dusp6 during early development in Xenopus laevis
(A) qRT-PCR of Dusp6 (both Dusp6.L and Dusp6.S) in the animal cap explants injected with Fgf8a mRNA or treated with SU5402. mRNA of Fgf8a or EGFP (as a control) (100 pg) was injected into all blastomeres at the four-cell stage. SU5402 (or DMSO) was applied from stage 9 to stage 15. The expression of Dusp6 was normalized to that of Ef1α. Statistical analysis was performed with unpaired two-tailed Student's t-test comparing the average of three biological replicates (∗p > 0.05, ∗∗∗p > 0.001). (B) Dusp6 expression decreases pERK1/2 levels. pERK1/2 and ERK1/2 were detected by western blotting in the animal cap explants injected with Fgf8a mRNA (100 pg) and/or Dusp6 mRNA (250 pg). Tubulin was employed as a loading control. (C–E) The expression of Dusp6 in Xenopus embryos. The expression in mid-gastrula was shown in (C; vegetal view, the anterior of embryos was positioned to the top). The dotted circle indicates the yolk plug (YP). The expression at the mid-neurula stage was shown in (D, anterior view; D’, dorsal view). The white arrowhead indicates mid-hind brain boundary (MHB). The dotted line in (D) indicates the position of the section shown in (D’’). The lateral view of the tailbud stage embryo was shown in (E). (F, G) Six1 and Fgf8a expression at the mid-neurula stage. The embryos were cut at the position of the dotted line in (F; anterior view) and (G; anterior view), and the sections were shown in (F’), (G’). (H–J) The expression of Dusp6, Six1 or Fgf8 (purple; NBT/BCIP) were double-stained with that of Sox3 (red; Fast Red) in the mid-gastrula stage embryos. Arrowheads indicate the expressed region colored by NBT/BCIP. Lines on the figures indicate the midline of embryos, and dashed line indicate the outline of Sox3 expression. Scale bars: 500 μm. OV, otic vesicle; BA, branchial arch; NP, neural plate; NNE, non-neural ectoderm; TB, tailbud.
Fig. 2. Dusp6 is necessary for the ectodermal patterning
(A) Schematic figure of the experiment in (B)–(E). mRNA or MO was unilaterally injected into two blastomeres of 4-cell stage embryos as indicated. (B, C) The expression of Six1FoxD3, Sox3 and Krt12.4 in the embryos unilaterally injected with Dusp6 (250 pg) mRNA (B) or Dusp6 MO (40 ng) (C) (with a tracer, membrane anchored GFP (memGFP) mRNA) at the 4-cell stage. The injected side, which was colored with red (immunostaining of memGFP), was represented by “inj” in the figures. Arrows or arrowheads denote the expansion or reduction of the expressing domain in comparison with the uninjected side, respectively. (D, E) Schematic representation of Dusp6 overexpression (B) or knockdown (C) experiments. (F) The expression of Six1, FoxD3 and Sox3 in the embryo unilaterally injected with dnDusp6 (2 ng) at the 4-cell stage. Scale bars: 500 μm. NC, neural crest; Epi, Epidermis.
Fig. 3. Dusp6 is necessary for appropriate NPB gene expression
(A) Zic1 and Pax3 expression at the mid-gastrula stage. Vegetal views, anterior toward the top. (B) Zic1 and Pax3 expression in the early-neurula stage embryos unilaterally injected with Dusp6 MO (40 ng) at the 4-cell stage. Arrow or arrowhead denotes the expansion or reduction of expressing domain in comparison with the uninjected side. (C) Otx2, Krox20 and Six1 expression in the mid-neurula stage embryos unilaterally injected with Dusp6 MO (40 ng) at the 4-cell stage. MO was injected into the animal pole of both dorsal and ventral blastomeres as Fig. 2A memGFP mRNA was co-injected with Dusp6 MO as a lineage tracer and detected by IHC (red). Scale bars: 500 μm.
Fig. 4. Dusp6 is expressed in the PPE in a FGF dependent manner at mid-neural stage
(A) Dusp6 expression in the mid-neurula stage embryos unilaterally injected with increasing dose of Fgf8a mRNA at the 4-cell stage. Low doses (12.5 and 25 pg) of Fgf8a decreased Dusp6 expression (white arrowhead), and high doses (50 pg and 100 pg) of Fgf8a suppressed Dusp6 expression not only in the injected side but also in the uninjected side (contralateral side: black arrowhead). The highest dose of Fgf8a induced ectopic Dusp6 expression (white arrow). Anterior view, the dorsal was positioned to the top. (B–C’) Dusp6 expression in the embryo into which FGF-soaked beads or control beads were implanted. An enlarged image of the dotted box region in (B) and (C) was shown in (B’) and (C’) respectively. The bead implanted area was represented by asterisk (around PPE region), and the ectopic expression was indicated by arrowhead. (D–E) FGF-soaked beads were implanted into epidermis (D) or neural plate (E) at mid-neurula stage. An enlarged image of (D) and (E) were shown in (D’) and (E’) which are indicated as dotted box. (D) and (D’) were shown in ventral view, anterior towards the top. (E) and (E’) were shown in anterior view. Lines on the embryos indicate the midline of embryos. Ectopic expression was observed in the vicinity of the beads (arrowhead). (F) Dusp6 expression in the embryos treated with SU5402 at varying concentration or DMSO (control). SU5402 or DMSO was applied from the stage 13 and the embryos fixed at the stage 15. Scale bars: 500 μm.
Supplementary Fig. 1 Expression pattern of Dusp6.S in X. laevis and X. tropicalis
(A) Dusp6.S expression in X. laevis at mid-neurula stage. Similar to Dusp6.L, it was expressed in PPE and anterior non-neural ectoderm. (B) Dusp6 expression in X. tropicalis at mid-neurula stage and tailbud stage. X. tropicalis Dusp6 was expressed in PPE and anterior non-neural ectoderm, similar to X. laevis Dusp6 genes. Additionally, faint expressions were observed in midbrain-hindbrain boundary (MHB), and rhombencephalon (R) at mid-neurula stage. At tailbud stage, X. tropicalis Dusp6 was expressed in otic vesicle (OV), branchial arch (BA) and tips of tailbud (TB), similar to that of Dusp6.L in X. laevis. Scale bars: 500 μm.
Supplementary Fig. 2 Phenotypes of Dusp6 knockdown and overexpression (A-D) Increasing doses of Dusp6 mRNA or MO were injected into the animal pole of dorsal blastomeres at the 4-cell stage. The phenotypes of Dusp6 overexpression (A) or knockdown (C) were classified by the eye formation: retina malformation was denoted as ‘slight’, and absence of eye was denoted as ‘severe’. These typical phenotypes were shown in (A) and (C). (B, D) Quantification of phenotypes indicated in (A) and (C). Total number of embryos was shown on the top of each bar. Scale bars: 500 μm.
Supplementary Fig. 3 Validation of Dusp6 MO
(A) Schematic diagram of Dusp6MO-GFP mRNA. the EGFP sequence was combined with the Dusp6 MO binding sequence. (B) The translation of EGFP was suppressed by Dusp6 MO. mRNA and/or MO were unilaterally injected into two blastomeres at the 4-cell stage and the specimens were observed at mid- gastrula stage. EGFP was observed in the embryos into which Dusp6MO-GFP or Dusp6MO-GFP + control MO was injected. We injected 500 pg Dusp6MO- GFP mRNA with/without 40 ng control MO or 40 ng Dusp6 MO. Scale bar: 1 mm. (C) Western blot analysis was performed using the embryos in (B). Lysates were isolated from the mid-gastrula embryos.
Supplementary Fig. 4 Validation of dnDusp6 activity
pERK1/2 and ERK1/2 in animal cap explants from the embryos into which was injected either EGFP (as a control) (2 ng), Dusp6.L (250 pg) or dnDusp6.L (2 ng) were detected by western blotting at stage 15.
Supplementary Fig. 5 Confirmation of effect of Dusp6 MO and Fgf8a mRNA on AP patterning
(A) Expression of Otx2, Krox20 and Six1 in the mid-neurula stage embryos unilaterally injected with Dusp6 MO (40ng) at the 8-cell stage. While Six1 expression was reduced, there was no obvious shift in Otx2 and Krox20 patterns in the along AP axis. (B) Expression patterns of AP markers (Otx2, Krox20, MyoD) and Dusp6 in embryos unilaterally injected at the 4-cell stage with 12.5 pg or 100 pg Fgf8a mRNA with memGFP as a lineage tracer (red). Otx2 expression decreased by Fgf8a mRNA injection (arrowhead). Krox20 and MyoD expression was shifted anteriorly (dashed lines). Dusp6 expression was diminished at the injected side (white arrowhead), compared to the uninjected side (black arrowhead) with a 12.5 pg injection, and ectopically induced at 100 pg injection (arrow). Only MyoD was showed in a dorsal view, posterior to the top. Scale bars: 500 μm.
Supplementary Fig. 6 FGF-soaked beads implantation affects the Six1 expression pattern in Dusp6-knockdowned embryos
(A, B) The expression pattern of Six1 in all the morphants (Dusp6 MO (A) or control MO (B) injected) into which FGF-soaked beads were implanted (corresponded to Fig. 5B). Beads were implanted into the vicinity of the prospective PPE region on the left side in the figures (right in the embryos) as in Figure 5B. The details were shown in Materials and Methods. Each image has been modified for normalization by rescaling the embryos to have the same height (A-P length) and width (left-right ends). (C) Delta Y at each position was measured by the difference between bead implanted side and the other. (D) Max distance of the Six1 pattern on bead implanted side was defined as the difference in AP axis direction from the anterior end of the notoplate to the most anterior point among a, b, or c. Delta of max distance was defined as the difference of the max distance at symmetrical positions on the left and right sides, and coefficient of variation (CV) was calculated for the delta of max distance. Statistical analysis for equality of variances was performed using Levene’s test ( p = 0.00086 < 0.05).
Supplementary Fig. 6 FGF-soaked beads implantation affects the Six1 expression pattern in Dusp6-knockdowned embryos
(A, B) The expression pattern of Six1 in all the morphants (Dusp6 MO (A) or control MO (B) injected) into which FGF-soaked beads were implanted (corresponded to Fig. 5B). Beads were implanted into the vicinity of the prospective PPE region on the left side in the figures (right in the embryos) as in Figure 5B. The details were shown in Materials and Methods. Each image has been modified for normalization by rescaling the embryos to have the same height (A-P length) and width (left-right ends). (C) Delta Y at each position was measured by the difference between bead implanted side and the other. (D) Max distance of the Six1 pattern on bead implanted side was defined as the difference in AP axis direction from the anterior end of the notoplate to the most anterior point among a, b, or c. Delta of max distance was defined as the difference of the max distance at symmetrical positions on the left and right sides, and coefficient of variation (CV) was calculated for the delta of max distance. Statistical analysis for equality of variances was performed using Levene’s test ( p = 0.00086 < 0.05).
