Phenotypes of Daam morphants. (A) Injection of a morpholino oligonucleotide (MO) against Daam1a induced a short bent tail (red arrowheads) and cyclopia (blue arrowhead) at 3 d after fertilization (dpf). (B) Injection of a MO against EphB4 resulted in similar defects with a short bent tail (red arrowheads) and cyclopia (blue arrowhead). (C) Injection of a Daam1b MO also induced a similar phenotype. (D) Coinjection of Daam1a- and Daam1b MOs resulted in more severe abnormalities, consisting of a short bent tail (red arrowheads) and cyclopia (blue arrowhead). (E) In contrast, phenotype induced by coinjection of Daam1a- and Daam2 MOs was similar to that induced by the Daam1a-MO single injection (A). (F) Likewise, coinjection of Daam1b- and Daam2 MOs resulted in the same defect as the Daam1b MO (C). These observations indicate that Daam1a and Daam1b function in a redundant manner. (G) Triple injection of Daam1a-, Daam1b-, and Daam2 MOs induced more severe effects, with loss of eyes (blue arrowhead) and a short bent tail (red arrowheads). Because injection of Daam2 MO alone did not induce any obvious alteration, the contribution of Daam2 to notochord development is small (data not shown). (H) Loss-of-function of Dynamin1 (Dnm1) results in the phenotype characteristic of abnormal CE cell movement, consisting of compressed somites (red arrowheads) and a short body axis. (I and J) At the 80% epiboly stage, distribution of mesoderm cells marked by ntl became wider and shorter (J) than the wild-type embryo (I). (K) Coinjection of MOs against EphB2 and EphB4 induced the CE defects in 93.0% of embryos (n = 44). Coinjection of an flh-mEphB2 plasmid, which drives expression of mouse EphB2 in the notochord, rescued the defects, which were observed in 45.7% of injected embryos (n = 35). Injection of flh-mEphB2 alone induced the defects in only 3.0% of embryos (n = 33).
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