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Fig. 1. Amino acid sequence alignments of dullard with related proteins obtained from GenBank. Dullard shares 92% sequence identity with both a
human protein and a mouse protein, 68% sequence identity with a fly protein, and 62% sequence identity with a nematode protein. Dullard also
shares 64% sequence identity with the C-terminal conserved domain ofNLI-IF at the C-terminal region (amino acids 64â236).
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Fig. 2. (A–I) Temporal and spatial expression patterns of dullard. (A)
RT-PCR analysis was performed at the various stages indicated above
the lanes. From egg to stage 35, similar levels oftranscript were present.
(B–I) Whole mount in situ hybridization analysis of dullard expression.
(B–D) The transcripts localized to the animal hemisphere of
the embryo before the gastrula stage. (B) Eight-cell stage, lateral view.
(C) Stage 8, lateral view. (D) Stage 10.5, ventro-lateral view. (E–G) As
gastrulation proceeded, expression became restricted to the neural
region. (E) Stage 12, lateral view. (F) Stage 15, dorsal view. (G) Stage
20, dorsal view. (H, I) Expression subsequently localized to the neural
tissues, branchial arches, and pronephroi at the tail-bud stages. (H)
Stage 28, lateral view. (I) Stage 33, lateral view. Animal pole is towards
the top (B–D) and anterior is to the left (E–I). The arrowhead in (D)
and arrow in (E) indicate the dorsal lip and yolk plug, respectively.
(J, K) Overexpression of dullard caused apoptosis. (J) Dullard mRNA
(100 pg) was injected in one blastomere of2-cell embryos. White
mottled aspects were observed (95%, n ¼ 40). (K) Embryonic death
caused by overexpression of dullard was rescued by co-injection of
hBcl-2 mRNA (200 pg) (5% ofembryos exhibited white mottled aspects,
n ¼ 40).
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Fig. 3. Antisense dullard morpholino oligonucleotide (dullard Mo) blocked dullard mRNA function and injection of the dullard Mo caused failure of
neural tube closure and disorganization ofneural tissue. (A) Dullard mRNA (200 pg), injected embryos show white mottled aspects, characteristic of
apoptosis. (B) This effect was blocked by co-injection of dullard Mo (40 ng). (C–Q) Dullard Mo (40 ng/embryo) was injected in dorsal animal
blastomeres of8-cell stage embryos and the embryos raised until stage 24 or 35. (C) Control embryo at stage 24. (D) Dullard Mo (40 ng), injected
embryo at stage 24 shows failure ofneural tube closure. (E, F) Transverse sections ofstage 24 control and dullard Mo-injected embryos, respectively.
In the dullard Mo-injected embryo, the neural tube fails to close while other structures are normal. (G) Control embryo at stage 35. (H) Dullard Mo
(40 ng), injected embryo at stage 35 shows a reduction ofhead development and poor axis formation. (I) These effects were rescued by co-injection of
d5n-dullard mRNA. (J–Q) Transverse sections ofthe control (J–M) or dullard Mo-injected (N–Q) embryo at stage 35 at the sites indicated with small
letters on the whole embryos. The transverse section through the forebrain of a dullard Mo-injected embryo compared to that ofa control embryo
shows the absence ofhead structures (J, N). The transverse section through the midbrain ofa dullard Mo-injected embryo compared to that ofa
control embryo shows no or poor eye formation and disorganization of the midbrain (K, O). The transverse sections through the hindbrain and
spinal cord ofthe dullard Mo-injected embryo compared to that ofa control embryo show disorganization ofbrain and neural tube, while other
structures around them appear unaffected (L, M, P, Q).
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Fig. 4. Microinjection of dullard Mo suppressed expression of neural markers. Control morpholino oligonucleotide (A, C, E, G, I, K) or dullard Mo
(B, D, F, H, J, L) (40 ng) together with lacZ mRNA (200 pg) as a tracer was injected into a dorsal animal blastomere of8-cell embryos. Embryos were
harvested at the early tail-bud stage (A–D) or the neurula stage (remaining panels) and analyzed by whole mount in situ hybridization with the
indicated probes. In the dullard Mo-injected side ofthe embryo, expression ofthese markers is downregulated (B, D, F, H, J, L), while no change is
observed in the control morpholino oligonucleotide-injected embryos (A, C, E, G, I, K). (A and B) lateral view, (C–F) dorsal view, (G–L) anterior
view.
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ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4, horizontal view, animal up.
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ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, horizontal view, animal up.
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ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.
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ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
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