XB-ART-6955Biochem Biophys Res Commun July 5, 2002; 295 (1): 85-91.
Molecular cloning and characterization of dullard: a novel gene required for neural development.
In a screen for genes expressed in neural tissues and pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of NLI-IF (Nuclear LIM Interactor-Interacting Factor), a protein whose function is not yet characterized. Dullard mRNA was maternally derived and localized to the animal hemisphere. At neurula stages, the expression was in neural regions and subsequently localized to neural tissues, branchial arches, and pronephroi. Using antisense morpholino oligonucleotide-mediated inhibition, we showed that dullard was required for neural development. The translational knock-down of dullard resulted in failure of neural tube development and the embryos consequently showed a reduction of head development. Expression of neural marker genes in dullard-inhibited embryos was also suppressed. These results suggest that dullard is necessary for neural development.
PubMed ID: 12083771
Article link: Biochem Biophys Res Commun
Genes referenced: ctdnep1 egr2 en2 gmnn irx1 ldb1 ncam1 otx2 sox15 tubb2b zeb2 zic1 zic3
Morpholinos: ctdnep1 MO1
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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 64–236).|
|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).|
|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).|
|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.|
|ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4, horizontal view, animal up.|
|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.|
|ctdnep1 ( CTD nuclear envelope phosphatase 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.|
|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.|
|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.|