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???displayArticle.abstract??? Neptune, a Krüppel-like transcription factor, is expressed in various regions of the developing Xenopus embryo and it has multiple functions in the process of development in various organs. In situ hybridization analysis showed that Neptune is expressed in the boundary region between neural and non-neural tissues at the neurula stage, but little is known about the function of Neptune in this region. Here, we examined the expression and function of Neptune in the neural plate border (NPB) in the Xenopus embryo. Depletion of Neptune protein in developing embryos by using antisense MO caused loss of the hatching gland and otic vesicle as well as malformation of neural crest-derived cranial cartilages and melanocytes. Neptune MO also suppressed the expression of hatching gland and neural crest markers such as he, snail2, sox9 and msx1 at the neurula stage. Subsequent experiments showed that Neptune is necessary and sufficient for the differentiation of hatching gland cells and that it is located downstream of pax3 in the signal regulating the differentiation of these cells. Thus, Neptune is a new member of hatching gland specifier and plays a physiological role in determination and specification of multiple lineages derived from the NPB region.
Fig. 1. Expression pattern of Neptune at the neurula stage determined by whole-mount in situ hybridization analysis. (A) Neptune is expressed at the border region of the neural plate at stage 15 (n=12). Two lines of positive area along the neural fold were characteristic of the neurula stage. (B–D) Double in situ hybridization of Neptune (BM purple) with sox2 (Fast-Red) (B and B’, n=10), Neptune with sox9 (C and C’, n=10) and Neptune with keratin (D and D’, n=10). Dissection of stained embryos at the levels indicated by the broken lines in B–D showed the expression of Neptune (arrows) at the boundary between sox2- and sox9-positive regions and at the boundary between sox9- and keratin-positive regions (B’–D’). (E–H) Cross-sections of stained embryos. (A–C) Dorsal view and anterior to the bottom. (D) Lateral view and anterior to the left. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2.
Depletion of Neptune leads to loss of hatching gland and neural crest derivatives. (A) Alcian blue staining of cranial cartilages. Embryos injected with NepMO (20 ng) at the right side were fixed at stage 45. Morphogenesis of cartilage was disturbed at the NepMO-injected side (arrowheads) (disturbed in 9/15). A control tadpole at stage 45 is also shown (disturbed in 0/15). (B) Two right side blastomeres of 4-cell stage embryos were injected with CoMO (20 ng) or NepMO (20 ng) along with β-gal mRNA (0.3 ng). These embryos were cultured until stage 37 and stained with Red-Gal. The number of melanocytes at the NepMO-injected side was reduced (arrowheads) compared with the number of melanocytes at the uninjected side. CoMO did not affect the differentiation of melanocytes. The lower panel shows the results of statistical analysis of numbers of melanocytes from 10 animals. (C) Whole-mount antibody staining of hatching gland enzyme. Embryos was injected with NepMO (20 ng) or mutated NepMO (5misMO) (20 ng) at the right side, fixed at stage 30, and stained with an antibody against hatching gland enzyme. Staining was lost in the head region at the injected side (arrowhead) (lost in 8/13). No effect was found in the embryo injected with 5misMO (lost in 0/13). (D) Whole-mount antibody staining of keratan sulphate (upper panels) and muscle actin (lower panels). NepMO (20 ng) was injected into two right blastomeres at the 4-cell stage. The otic vesicle (arrowhead) disappeared at the NepMO-injected side (disappeared in 7/7). (E) Transverse section of the NepMO-injected tadpole at stage 37. The otic vesicle (ov) was lost at the injected side. Differentiation of the neural tube (nt) and notochord (n) was not affected
Fig. 3.
Loss of Neptune function suppresses the expression of hatching gland and NC markers. (A–P) Embryos were injected with β-gal alone (0.3 ng) (A, C, E, G, I, K, M, O) or NepMO (40 ng) and β-gal (0.3 ng) (B, D, F, H, J, L, N, P) in two right blastomeres at the 4-cell stage. At stages15–16, embryos were stained with Red-Gal and analyzed for the expression of tissue-specific markers by whole-mount in situ hybridization. In NepMO-injected embryos, expression of he (B), msx1 (D), sox9 (F), snail2 (H) was greatly reduced at the injected side (reduced in 26/29 for he, 24/30 for msx1, 70/90 for sox9 and 31/45 for snail2), but expression of pax3 was less affected (J) (expanded laterally in 26/32). The expression of sox2 was expanded laterally (L) (expanded laterally in 64/125) and, consistent with this observation, the expression of keratin at the midline was lost (N) (lost in 37/38). The expression of myoD was not affected by NepMO (P) (affected in 0/13). (Q–T) Dissected embryos that had been injected with NepMO and β-gal (R, T) or β-gal alone (Q, S). Expression of sox9 (Q, R) and sox2 (S, T) is shown. Expression of sox9 was suppressed at the injected side (R, left on the panel), whereas expression of sox2 was expanded at the injected side (T, left on the panel). The broken line indicates the midline. Arrowheads in S and T indicate the edge of sox2 expression.
Fig. 4.
Expansion of sox2 expression induced by NepMO is rescued by coinjection of Neptune-GR. Embryos were injected into two right blastomeres at the 4-cell stage with reagents and fixed at the neurula stage (stages 15–16). (A–D) Representative pattern of sox2 expression in embryos injected with β-gal (0.3 ng) (A), NepMO (40 ng) (B), or a combination of both NepMO (40 ng) and Δ5’UTR-Neptune-GR (1 ng) (C, D). β-gal (0.3 ng) was coinjected as a lineage tracer and stained with Red-Gal. Dexamethasone (DEX; 1 μM) was added to the culture medium at stage 12 (D). NepMO-injected embryos showed expanded expression of sox2 (B). Embryos injected with Δ5’UTR-Neptune-GR followed by treatment with DEX at stage 12 showed normal expression of sox2 and recovery of neural fold formation (D). Injected side is to the left (as shown by red staining). (E) Frequency distribution of phenotypes in each group of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5.
Loss of he expression by NepMO treatment is rescued by coinjection of Neptune-GR. (A–E) Embryos were injected into two right blastomeres at the 4-cell stage with β-gal (0.3 ng) alone (A), NepMO (20 ng) (B), Δ5’UTR-Neptune-GR (1 ng) (C), or a combination of both NepMO (20 ng) and Δ5’UTR-Neptune-GR (1 ng) (D, E). DEX (1 μM) was added to the culture medium at stage 12 (C, E). At stages 25–26, embryos were stained with Red-Gal and analyzed for the expression of he by whole-mount in situ hybridization. Expression of he was lost at the NepMO-injected side (B). Embryos injected with NepMO and Δ5’UTR-Neptune-GR followed by treatment with DEX at stage 12 showed recovery of he expression (E). (F) Frequency distribution of phenotypes in each group of experiments.
Fig. 6.
Neptune mediates the activity of pax3 for induction of hatching gland. (A–C) Embryos were injected into two right blastomeres at the 4-cell stage with Neptune-GR (1 ng) (B) or pax3 (1 ng) (C). DEX (1 μM) was added to the culture medium at stage 12 (B). Embryos were fixed at stage 30 and were stained with an antibody against hatching gland enzyme. Note that both Neptune and pax3 can ectopically induce the expression of HE (arrowheads in B and C) (increased in 15/16 by Neptune and 11/12 by pax3). (D–G) Embryos were injected with β-gal (0.3 ng) alone (D, F) or β-gal and pax3 (1 ng) (E, G) as described above. Embryos were fixed at stage 17, stained with Red-Gal, and analyzed for the expression of he (D, E) or Neptune (F, G) by whole-mount in situ hybridization, indicating that pax3 can induce the expression of he and Neptune at the injected side (arrowheads in E and G) (increased in 19/27 for he and 21/37 for Neptune). (H–J) Embryos were injected with pax3 and β-gal (H) or pax3 and β-gal together with NepMO (20 ng) (I) or 5misMO (20 ng) (J). Embryos were fixed at stage 17, stained with Red-Gal, and analyzed for the expression of he. Note that pax3-dependent induction of he was completely suppressed by the simultaneous injection of NepMO (arrowheads in I). he expression was increased in 23/31 embryos by pax3 injection but reduced in 23/28 embryos by pax3 and NepMO injection.
Fig. 7.
Loss of sox9 expression by NepMO treatment is rescued by coinjection of msx1. Embryos were injected into two right blastomeres at the 4-cell stage with reagents and fixed at the neurula stage (stage 15–16). (A–D) Representative pattern of sox9 expression in embryos injected with β-gal alone (0.3 ng) (A), β-gal with NepMO (40 ng) (B), β-gal with msx1 (1 ng) (C), or β-gal with NepMO (40 ng) and msx1 (1 ng) (D). Embryos were stained with Red-Gal to show the injected area. NepMO-injected embryos lost the expression of sox9 (B), and coinjection of NepMO and msx1 recovered the expression of sox9 (D). (E) Frequency distribution of phenotypes in each group of experiments.
klf17 (Kruppel-like factor 17) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior down.
