Huang et al., 2019 - Structural and functional studies of TBC1D23 C-terminal domain provide a link between endosomal trafficking and PCH. Proceedings of the National Academy of Sciences of the United States of America   116(45):22598-22608 Full text @ Proc. Natl. Acad. Sci. USA

Fig. 1

The TBC1D23 C terminus is critical for Zebrafish neuronal development and brain growth. (A) HuC (green) expression in Tg [HuC: GFP] transgenic zebrafish. WT, wild type; control, control MO injection; MO1, MO1 injection; MO1+FL, MO1 and full-length human TBC1D23 mRNA coinjection; MO1+P1, MO1 and P1 mRNA coinjection; MO1+P2, MO1 and P2 mRNA coinjection. P1 and P2 stand for 2 TBC1D23 mutations found in patients. P1 is a 2-base pair deletion (c.1475_1476delTG, NM_001199198.2) in the TBC1D23 gene, resulting in a frameshift, and a protein truncated the C terminus (Val492GlyfsTer8). P2 skips the exon 16, and encodes a truncated protein (His534TrpfsTer36). All injections are performed at one-cell stage of the development. (Top) Lateral views; (Bottom) dorsal views. (B) Classification of zebrafish embryos based on the expression level of HuC (elavl3) at 48 hpf. C1, normal; C2, moderately decreased; C3, significantly lost. (Top) Lateral views; (Bottom) dorsal views. (C) The percentage of embryos in each class upon injection of MO1 or coinjection of mRNAs. Experiments were at least triplicated for all zebrafish experiments, and n stands for the number of embryos used for statistical analysis. (D) Immunofluorescent staining with a 2-photon confocal microscope showing the effects on HuC (green) and DAPI (blue) of deleting TBC1D23. DAPI was used to label the entire head structure of zebrafish. Dorsal views. (E) Semi-quantitative RT-PCR analysis of the transcription level of HuC. Mean ± SEM; n = 3; ***P < 0.001; **P < 0.01; ns, not significant. P values were calculated using one-way ANOVA, Tukey’s multiple comparisons test, throughout the paper unless otherwise indicated. (F) Morphology of CaP axons from embryos at 48 hpf that were injected with MO1 and/or different mRNA. All injections are performed at one-cell stage of the Tg [hb9: GFP]ml2 transgenic zebrafish embryos. Arrows indicate abnormal branches. Lateral views and enlarged views are shown. Rectangles in Top are shown in Bottom. (G) Statistical results of the branch number of CaP axons in embryos were treated as in F. For each group, 40 axons from 8 Tg [hb9: GFP]ml2 transgenic zebrafish embryos are scored. Experiments were repeated 3 times. ***P < 0.001.


Fig. 6

Mutations of TBC1D23 in phosphoinositide- and FAM21-binding sites lead to abnormal motor neuronal development in zebrafish. (A) Expression of HuC (elavl3) in zebrafish at 48 hpf; control, control MO injection; MO1, MO1 injection; MO1+KR, MO1 and TBC1D23 K585D/R606D (KR) mutant mRNA coinjection; MO1+3K, MO1 and TBC1D23 K632E/K633E/K634E (3K) mutant mRNA coinjection. All injections are performed at one-cell stage of the development. (Left) Lateral view; (Right) dorsal view. (B) Whole-mount immunofluorescent staining of HuC (green) and DAPI (blue) showing the effects of the KR and 3K mutants. Dorsal views are shown. (C) Semi-quantitative RT-PCR analysis of the transcription level of HuC. Mean ± SEM; n = 3; ***P < 0.001; *P < 0.05. (D) Morphology of CaP axons from embryos at 48 hpf that were injected MO1 and/or different mRNA. All injections were performed at one-cell stage of the Tg [hb9: GFP]ml2 transgenic zebrafish embryos. Arrows indicate abnormal branches. (Left) Lateral views and (Right) enlarged views of rectangles at Left. (E) Statistical results of the branch number of CaP axons in embryos treated as in D. For the KO1+KR group, 48 axons from 10 different Tg [hb9: GFP]ml2 transgenic zebrafish embryos were used for analysis; for the rest of the groups, 30 axons from 6 embryos were used. ***P < 0.001; **P < 0.01.


EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage: Long-pec
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Long-pec

Fig. S1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage: Long-pec

Fig. S2 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Protruding-mouth
Acknowledgments:
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Proc. Natl. Acad. Sci. USA