Fig. 5. Krm is required for anterior CNS formation during Xenopus embryogenesis. (A) Krm morpholino oligonucleotides (Mo) act specifically to inhibit translation of their cognate cDNA construct when overexpressed in embryos. mRNA (750pg of both C-terminal V5 tagged krm2 and C-terminal HA tagged krm1) was co-injected equatorially into both blastomeres of two-cell stage embryos. The same embryos were then injected with 2.5 ng of the indicated morpholinos in all four vegetal blastomeres at the eight-cell stage and harvested at stage 11. Tagged Krm proteins were visualised by western blot analysis using either anti-V5 IgG (Krm2, top panel) or anti-HA (Krm 1, middle panel). A crossreacting protein band from the anti-HA western is shown as a loading control (bottom panel). (B-D) Krm proteins are required for normal head formation. All four animal blastomeres of eight-cell stage embryos were injected with either 5 ng of control Mo (B), 2.5 ng each of krm1 + krm2 (krm1/2) Mo (C), or co-injected with krm1/2 Mo and 100pg krm2 DNA (D) and allowed to develop for 4 days. (B) Embryos injected with control Mo show no abnormalities. (C) Embryos injected with krm1/2 Mo show microcephaly and slight shortening of the trunk/tail region (85%, n=400). (D) Rescue of krm1/2 Mo phenotype by co-injection of pCS-Xkrm2 DNA. Rescue, similar to that shown in D, was seen in 25% (n=300) of co-injected embryos. (E-H) Krm is required for formation of anterior neural tissue. (E,F) bf1 in situ hybridisation of stage 25 embryos marks clear reduction of forebrain tissue (red asterisk) in krm1/2 Mo injected embryo. Frontal views of head region are included at centre-top of panels. (G,H) Double bf1/en2 in situ hybridisation of stage 16 embryos injected in one dorsal blastomere at the four-cell stage with lacZ mRNA (used as tracer) and either 5 ng control Mo (G) or krm1/2 Mo (H). A reduction of the bf1 expression, but normal en2 expression was seen in 40% (n=60) of krm1/2 Mo-injected embryos. (I-T) Krm and Dkk1 cooperate in head formation. Embryos were injected in all four animal blastomeres at the eight-cell stage with 5 ng control Mo (I,M) or 2.5 ng each of krm1/2 Mo (J,N and L,P). At stage 9, the same embryos were injected with either PBS (I,M and J,N) or 100 ng anti-Dkk1 antibody (K,O and L,P) into the blastocoel and allowed to develop for 3 days. Note the similarity in phenotypes for krm1/2 Mo and anti-Dkk antibody injections (compare especially N and O with M) and their synergy when combined (L,P). (M-P) The corresponding frontal views of embryos shown laterally in I-L. No headless embryos (n=50-110) were observed in I-K, but 40% (n=70) were headless in L. (Q-S) bf1 in situ hybridisation of late neurula embryos injected as described above with control Mo (Q), krm1/2 Mo (R), anti-Dkk1 (S) and krm1/2 Mo + anti-Dkk1 (T). Compared with the controls (Q), reduction/loss of bf1 expression domain was seen in 40/0% (R, n=200), 15/0% (S, n=35) and 40/60% (T, n=25) of embryos.

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