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Fig. 2.
PN1 promotes anterior development, suppresses mesoderm and reduces neuronal differentiation. (A) Uninjected tadpole embryo. (B) Injection of PN1 mRNA induces enlargement of head structures and coloboma (arrowhead in inset). (C) Co-injection of PN1 and HtrA1 mRNA restores normal development. (D-K) Whole-mount in situ hybridization of post-neurula embryos in anterior view. PN1 causes enlargement of the Foxg1, En2, Fgf8 and Nkx2-5 expression domains. Rax expression is not split into bilateral domains. (L-O) Xbra expression in early gastrulae, lateral view. A single marginal injection of PN1 strongly reduces Xbra expression (M). PN1 and HtrA1 partially revert this effect (N). PN1pm mRNA causes only mild or no reduction of Xbra expression (O). (P,Q) Ventral view of neurulae. PN1 expands anterior Sizzled expression. (R-X,BB) Dorsal view of neurula embryos. A single injection of PN1 causes reduction and posteriorward retraction of N-tubulin (arrowheads in S), reduction of Papc and expansion of Otx2 expression (brackets in W) on the targeted right side. PN1 and HtrA1 rescue these effects (T,BB). PN1pm does not affect these markers (U,X). (Y-AA) Injection of 15 ng HtrA1-MO, XFD mRNA or Dkk1 mRNA also causes anteriorization. (CC) Fgf4 mRNA rescues anteriorization by PN1. (DD) pCS2-Wnt3a (Wnt3a-DNA) reverts PN1-induced Otx2 expansion, but not Papc reduction. Total mRNA amounts were: PN1 constructs, 4 ng (1 ng in W,X,BB-DD, 16 ng in E,K,Q); HtrA1, 100 pg; XFD, 80 pg; Dkk1, 8 pg; Fgf4, 0.3 pg. Indicated phenotypes were shown by: B, 44/56; C, 30/30; E, 19/21; G, 16/16; I, 13/15; K, 14/17; M, 71/73; N, 18/32; O, 23/32; Q, 9/19; S, 41/42; T, 16/19; U, 42/42; W, 57/62; X, 31/39; Y, 20/24; Z, 27/31; AA, 59/60; BB, 19/23; CC, 19/24; DD, 15/16. |
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Fig. 4.
Redundant functions of PN1.a and PN1.b in head and primary axis development. (A) Three antisense morpholino oligonucleotides (MOs) target the translation initiation sites of the PN1.a and PN1.b homeologs. (B) Immunoblot analysis of Xenopus gastrula embryos. PN1.a-MO1+2 and PN1.b-MO, but not standard control MO (co-MO), inhibit translation of injected PN1.a-FLAG and PN1.b-FLAG mRNAs (each 800 pg), respectively. Protein synthesis from non-targeted FLAG-PN1 mRNA (800 pg) is not affected. WB, western blot. αTubulin provides a loading control. (C) PN1-MO blocks endogenous PN1 protein expression (arrowhead). The asterisk marks a non-specific band. Ponceau Red staining shows equal protein loading. (D) Co-MO-injected tadpole. (E-G) Microinjection of either PN1.a-MO1+2 or PN1.b-MO causes microcephaly. A combination of all three MOs (designated PN1-MO) results in severe reduction of head and shortening of tail structures. (H-Q) PN1-MO induces depletion of En2 and reduction of Foxg1, Rax and Nkx2-5 expression. Fgf8 and Ism expression is severely reduced at the midbrain-hindbrain boundary (arrowheads). (R,S) Reduction of anterior and expansion of posterior Sizzled expression in PN1-depleted embryo. Indicated phenotypes were shown by: D, 29/31; E, 125/161; F, 102/134; G, 34/38; H, 71/77; I, 26/28; J, 45/45; K, 60/67; L, 40/45; M, 61/66; N, 16/18; O, 25/29; P, 40/40; Q, 25/25; R, 53/54; S, 33/39. |
