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Members of the plexin protein family are known regulators of axon guidance, but recent data indicate that they have broader functions in the regulation of embryonic morphogenesis. Here we provide further evidence of this by showing that PlexinA1 is expressed in Xenopus neural crest cells and is required for their migration. PlexinA1 expression is detected in migrating cranial neural crest cells and knockdown of PlexinA1 expression using Morpholino oligonucleotides inhibits neural crest migration. PlexinA1 likely affects neural crest migration by interaction with PTK7, a regulator of planar cell polarity that is required for neural crest migration. PlexinA1 and PTK7 interact in immunoprecipitation assays and show phenotypic interaction in co-injection experiments. Considering that plexins and PTK7 have been shown to genetically interact in Drosophila axon guidance and chick cardiac morphogenesis, our data suggest that this interaction is evolutionary conserved and may be relevant for a broad range of morphogenetic events including the migration of neural crest cells in Xenopus laevis.
Fig. 1.
In situ hybridization shows that PlexinA1 is expressed in Xenopus laevis NC cells. (A–C) PlexinA1 is expressed in the animal hemisphere of a one-cell stage embryo (A), a blastula stage 8 embryo (B) and a gastrula stage 10.5 embryo (C). Sense controls are shown on the left. (D, E) PlexinA1 expression is detected in the area of the closing neural tube (white arrow) and in premigratory NC cells at neurula stage 16 (D) and neurula stage 23 (E). (F) At tadpole stages PlexinA1 is expressed in the migrating NC cells, the eye, the pronephros and the somites. Abbreviations: nc, neural crest; pn, premigratory NC cells; p, pronephros; s, somites.
Fig. 2.
Overexpression of PlexinA1 leads to a reduction in migrating NC cells at neurula stages. (A) Xenopus embryos were injected with 500 pg PlexinA1 RNA and 100 pg lacZ RNA as a lineage tracer in one blastomere at the two-cell stage. Twist in situ hybridization was used to analyze NC migration at neurula stage 20 or tailbud stage 28. LacZ-injected control embryos are shown in the upper panel, PlexinA1 injected embryos in the lower panel. “+†marks the injected lacZ-positive (blue) side and “−†the uninjected side, black arrow head indicates NC migration defects. The left panels show an anterior view of a neurula stage embryo, the right panels the head region of injected tailbud stage embryos. (B) Graph summarizing seven independent injection experiments (three experiments did not include injections of 250 pg PlexinA1 RNA). Embryos were injected either with 100 pg lacZ RNA alone or in combination with 250 pg or 500 pg PlexinA1 RNA. Number of injected embryos is indicated for each column. (∗) NC migration defects were significantly different compared to lacZ injected controls (p < 0.01 in a student t-test) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 3.
PlexinA1 loss of function inhibits NC migration. (A–D) Embryos were injected in one blastomere at the two-cell stage with the indicated constructs in combination with the lineage tracer lacZ. RFP RNA was used to equalize the concentration of injected RNA. NC migration was analyzed by twist in situ hybridization at neurula stage 20–22. The injected lacZ-positive (blue) side is shown on the right. (A) Embryo injected with 20 ng control MO (co MO) shows normal NC migration. (B) Embryo injected with 20 ng PlexinA1 MOs showing an inhibition of NC migration (white arrow). (C) Embryo injected with control MO and 300 pg rPlxA1 RNA showing an inhibition of NC migration (white arrow). (D) Embryo with normal NC migration after co-injection of 300 pg rPlxA1 RNA together with PlexinA1 MOs. (E, F) Tailbud stage 28 embryo, injected side is shown in the right panel. (E) Embryo that was injected with 20 ng control MO shows normal NC migration. (F) Injection of 20 ng PlexinA1 MOs cause an inhibition of NC migration (white arrow). (G) Western Blot showing that the expression of an HA-tagged rescue construct, which lacks both MO binding sites (HA-rPlxA1), is not affected by the PlexinA1 MOs or the control MO (right lanes). In contrast, the expression of a construct containing the PlexinA1 MO2 binding site (HA-PlxA1) is inhibited by the PlexinA1 MOs (left lane). Embryos were injected in one blastomere at the one-cell stage with the indicated constructs and lysed at gastrula stage 10.5. The expression of the HA-tagged plexin constructs was detected by HA antibody staining (upper panel). Actin expression was monitored as a control (lower panel). (H) Graph summarizing five independent experiments using 300 pg rPlxA1 RNA (two experiments did not include injections of 500 pg rPlxA1 RNA). The percentage of NC migration defects caused by the PlexinA1 MOs of individual experiments was set to 1. Embryos were injected with the indicated constructs, MOs at a concentration of 20 ng. NC migration defects were scored at stage 20–22. (∗) Injection of 300 pg of the rescue constructs significantly improved the NC migration defects caused by the PlexinA1 MOs (p < 0.005 in a student t-test) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 4.
PlexinA1 and PTK7 interact in immunoprecipitation and phenotypic assays. (A) Full length HA-tagged PlexinA1 (PlxA1-HA) precipitates myc-tagged PTK7 (PTK7-myc). Upper panels show the immunoprecipitated proteins, the lower panels the Xenopus lysates. Embryos were injected with 800 pg PTK7-myc RNA and 1 ng PlexA1-HA RNA. 50 Embryos were lysed at gastrula stage and used for immunoprecipitation. Injected constructs and antibodies used for Western blotting are indicated. (B–F) Co-expression of PlexinA1 and PTK7 leads to an increase in NC migration defects. Xenopus embryos were injected with the respective RNA construct in combination with 75 pg lacZ RNA in one blastomere at the two-cell stage. RFP RNA was co-injected to equalize injected RNA concentrations. NC migration was analyzed by twist in situ hybridization at neurula stage 22. (B) Graph summarizing the percentage of NC migration defects of three independent injection experiments. Number of injected embryos is indicated for each column. (∗) Percentage of NC migration defects caused by co-injection of PlxA1 and PTK7 is significantly different from the PlxA1 injected embryos in a student t-test (p < 0.05). (C–F) Twist in situ hybridization of injected neurula stage 20–22 embryos, the injected lacZ-positive (blue) side is shown on the right. (C) Embryo injected with 1 ng RFP RNA shows normal NC migration. (D) Embryo injected with 500 pg PTK7 RNA and 500 pg RFP RNA showing normal NC migration. (E) Embryo injected with 500 pg PlexinA1 RNA and 500 pg RFP RNA. NC migration is affected (white arrow). (F) NC migration is inhibited in an embryo co-injected with 500 pg PlexinA1 and 500 pg PTK7 RNA. NC cells are induced, but fail to migrate (white arrow) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
plxna1 (plexin A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
