Morpholino-induced knockdown of fgf8 efficiently phenocopies the Acerebellar (ace) phenotype
Morpholino-induced knockdown of fgf8 efficiently phenocopies the Acerebellar (ace) phenotype
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DOI：
10.1002/gene.1054
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发表时间：
2001-07-01
期刊：
影响因子：
1.5
通讯作者：
Brand, M
中科院分区：
文献类型：
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作者：
Araki, I;Brand, M
纯合小脑 (ace) 胚胎缺乏小脑和中脑-后脑边界 (MHB) 组织者，并且在前脑和心脏发育方面存在缺陷 (Brand 等，1996；Picker 等，1999；Reifers 等，1998；Shanmugalingam 等(2000)；Raible 和 Brand，2001；Araki 和Brand，未发表的数据），表明这可能是因为Fgfs之间的功能冗余。由于吗啉代可能有助于解决此类问题，因此我们试图通过注射针对 fgf8 (MO-fgf8) 的吗啉代来对因 Ace/Fgf8 缺失而导致的已知缺陷进行表型复制。尽管 ace 表型很复杂，我们发现 MO-fgf8 在 MHB、前脑和心脏发育中有效且一致地复制了 ace 突变体。我们设计了针对 fgf8 的反义吗啉代，覆盖翻译起始密码子（图 1J）。注射到一到八细胞阶段的野生型胚胎后，我们发现 MO-fgf8 有效地复制了 ace 表型。我们使用 0.5 至 4 g/l 的 MO-fgf8 进行注射（表 1），每个胚胎可输送 1.6 至 12.6 ng 的 MO-fgf8。从形态学上看，注射 1-4 g/l 的 24 小时阶段的胚胎缺乏小脑和 MHB 组织者，同一阶段的 ace 突变体也是如此（图 1A-I）。具有四个错配碱基对的吗啉代与 fgf8（图 1J 中的对照 MO）没有效果，在相同浓度下也没有显示任何非特异性效果（表 1）。为了检查 ace 表型的模仿程度，我们用 pax2 探针对注射的胚胎进行了染色。图 1 揭示了 Fgf8 发挥功能的几种组织。帕克斯2。 MHB 上的 1 表达最初在 ace 突变体中是正常的，但没有得到适当维持（Reifers 等，1998；Lun 和 Brand，1998）。 24小时时，注射MO-fgf8的胚胎要么缺乏pax2。 1 在 MHB 完全表达，或者减少到一个小的背侧斑块，如同一阶段的 ace 突变体中所见（图 1D-I 且未显示；Reifers 等人，1998）。由于 ace 突变体在前脑和心脏发育中也表现出缺陷，我们用分子标记分析了 MO-fgf8 注射是否也可以在这些组织中对 ace 突变体进行表型复制。受精后十小时，注射的胚胎减少并扰乱了 emx1（一种早期端脑标志物）的表达，如 ace 突变体（图 1K-M；Shanmugalingam 等，2000）。类似地，24小时时pax2的表达。视柄中的 1 减少，视交叉减少或缺失（未显示），如之前在 ace 突变体中发现的那样（Shanmugalingam 等，2000）。注射的胚胎用 nkx2 染色。 5 是心脏原基的早期标志物，显示 nkx2 下调。 5，正如在 ace 突变体中观察到的那样（图 1N-P；Reifers 等人，2000a）。我们没有检测到 MO-fgf8 的任何非特异性作用。我们得出结论，MO-fgf8 注射有效地复制了 MHB、端脑、视柄和心脏发育中已知的小脑功能丧失表型，从而验证了该方法的有用性。因为注射吗啉被认为可以阻止翻译（对于 fgf8，我们认为这是正确的，但尚未测试），这些发现也支持（Reifers 等人，1998）ace 是无效等位基因。为了进一步测试这个概念，我们检查了是否可以通过注射 Mo-fgf8 来增强纯合小脑突变体的表型。形态学上和检查 pax2 之后。 1 在尾芽、二体节、五体节和 24 小时阶段的注射胚胎中表达，我们没有观察到同一窝中的 ace 纯合子和它们的野生型兄弟姐妹之间的差异（表 1 和未显示），表明 ace 表型无法进一步增强，因此很可能......
Homozygous acerebellar (ace) embryos lack their cerebellum and the midbrain–hindbrain boundary (MHB) organizer, and in addition have defects in forebrain and heart development (Brand et al., 1996; Picker et al., 1999; Reifers et al., 1998, Shanmugalingam et al.(2000); Raible and Brand, 2001; Araki and Brand, unpublished data), suggesting that this may be because of functional redundancy between Fgfs. Because morpholinos might help to resolve such issues, we sought to phenocopy the known defects caused by absence of Ace/Fgf8 through injection of a morpholino against fgf8 (MO-fgf8). In spite of the complexity of the ace phenotype, we find that MO-fgf8 efficiently and uniformly phenocopies the ace mutant in MHB, forebrain, and heart development. We designed an antisense morpholino against fgf8, covering the translational start codon (Fig. 1J). After injections into one-to eight-cell-stage wild-type embryos, we find that MO-fgf8 effectively phenocopies the ace phenotype. We used between 0.5 and 4 g/l of MO-fgf8 for the injection (Table 1), which delivers between 1.6 and 12.6 ng of MO-fgf8 per embryo. Morphologically, embryos at the 24-h stage injected with 1–4 g/l lacked the cerebellum and the MHB organizer, as do ace mutants at the same stage (Fig. 1A–I). A morpholino with four mismatched base pairs against fgf8 (control MO in Fig. 1J) had no effect, nor did it show any nonspecific effects at the same concentration (Table 1). To examine how closely the ace phenotype is mimicked, we stained the injected embryos with a probe for pax2. 1 which reveals several of the tissues where Fgf8 functions. pax2. 1 expression at the MHB is initially normal in ace mutants, but is not properly maintained (Reifers et al., 1998; Lun and Brand, 1998). At 24 h, MO-fgf8 injected embryos either lacked pax2. 1 expression at the MHB completely, or it was reduced to a small dorsal patch, as seen in ace mutants at the same stage (Fig. 1D–I and not shown; Reifers et al., 1998). Because ace mutants show defects also in forebrain and heart development, we analyzed with molecular markers whether MO-fgf8 injection can phenocopy the ace mutant also in these tissues. Ten hours after fertilization, injected embryos have reduced and perturbed expression of emx1, an early telencephalic marker, as in ace mutants (Fig. 1K–M; Shanmugalingam et al., 2000). Similarly, at 24 h expression of pax2. 1 in the optic stalk is reduced, and in the optic chiasm is reduced or missing (not shown), as found previously for ace mutants (Shanmugalingam et al., 2000). Injected embryos stained with nkx2. 5, an early marker for heart primordium, showed downregulation of nkx2. 5, as is observed in ace mutants (Fig. 1N–P; Reifers et al., 2000a). We did not detect any nonspecific effect of MO-fgf8. We conclude that MO-fgf8 injection efficiently phenocopies the known loss-of-function phenotype of acerebellar in MHB, telencephalon, optic stalk, and heart development, thus validating the usefulness of this method. Because morpholino injection is thought to prevent translation (which we assume to be true, but have not tested, for fgf8), these findings also support (Reifers et al., 1998) that ace is a null allele. To further test this notion, we examined whether the phenotype of homozygous acerebellar mutants can be enhanced by Mo-fgf8 injection. Morphologically and after examining pax2. 1 expression in injected embryos at the tailbud, two-somite, five-somite, and 24-h stage, we did not observe a difference between ace homozygotes and their wild-type siblings in the same clutch (Table 1 and not shown), showing that the ace phenotype cannot be further enhanced and therefore most likely …