Wdr68 requires nuclear access for craniofacial development

Abstract

Wdr68 is a highly conserved scaffolding protein required for craniofacial development and left-right asymmetry. A Ras-Map3k-Wdr68-Dyrk1 signaling relay may mediate these and other diverse signaling events important in development and disease. While the sub-cellular localization of Wdr68 has been shown to be dependent on that of its interaction partners, it is not clear where Wdr68 activity is required during development. Here we show that while a GFP-Wdr68 fusion functionally substituted for craniofacial development in the zebrafish, that a Nuclear Export Signal (NES) fusion protein (GFPNESWdr68) failed to support craniofacial development. As control for NES activity, we show that while GFP-Wdr68 exhibited a pan-cellular distribution in C2C12 cells, the GFPNESWdr68 fusion predominantly localized to the cell cytoplasm, as expected. Interestingly, while GFP-Wdr68 and RFP-Dyrk1a co-localized to the cell nucleus as expected based on the known sub-cellular localization for Dyrk1a, we found that the GFPNESWdr68 fusion redistributed RFP-Dyrk1a to the cell cytoplasm potentially disconnecting the Ras/Dyrk1 signal relay from further downstream targets. Consistent with a nuclear role in gene regulation, we also found that while a transcriptional activation domain fusion, CebpFlagWdr68, functionally substituted for endogenous Wdr68 for craniofacial development, that a transcriptional repression domain fusion, MadFlagWdr68, failed to support craniofacial development. Dyrk1b is required for myogenin (myog) expression in differentiating mouse C2C12 cells and here we report that wdr68 is also important for myog expression in differentiating C2C12 cells. Using a C2C12 cell myog promoter-reporter system, we found that Wdr68 overexpression increased reporter activity while moderate expression levels of MadFlagWdr68 interfered with reporter activity. Taken together, these findings support a nuclear role for Wdr68-containing complexes.

Figure 1. Nuclear access is required for Wdr68 function in craniofacial development.

A–H) Alcian blue stained animals. A, C, E, G) Ventral view with the Meckel’s (M), palatoquadrate (PQ) and ceratohyal (CH) labeled in red. B, D, F, H) Lateral view. A, B) Wildtype control-MO (ctrl) injected animals. C, D) Severe craniofacial phenotype of wdr68-MO injected animals co-injected with GFP mRNA (MO+GFP) that fails to rescue the craniofacial defects. E, F) Rescue of the craniofacial defects of wdr68-MO animals co-injected with GFP-Wdr68 mRNA (MO+GW). G, H) Severe craniofacial defects of wdr68-MO animals co-injected with GFPNESWdr68 mRNA (MO+GNESW) that fails to rescue the craniofacial defects. I) Graph summarizing the results of three injection trials. In aggregate, n = 50/50 (100%) for control animals, n = 47/257 (18%) for MO+GFP animals, n = 212/350 (61%) for MO+GW animals, and n = 71/385 (18%) for MO+GNESW animals.

Figure 2. The GFPNESWdr68 fusion redistributes interaction partner Dyrk1a from the nucleus to the cytoplasm in C2C12 cells.

A, E, I, M, Q, U) DAPI stained cell nuclei. B, F, J, N, R, V) GFP fusion proteins. C, G, K, O, S, W) mRFP1 fusion proteins. D, H, L, P, T, X) Overlays of blue, green and red channels. A–D) pan-cellular distribution of GFPWdr68 (GW) alone. E–H) Cytoplasmic restriction of GFPNESWdr68 (GNESW) alone. I–L) Redistribution of GW to the cell nucleus when co-expressed with mRFP1-Dyrk1a. M–P) Redistribution of mRFP1-Dyrk1a when co-expressed with GNESW. Q–T) Pan-cellular distribution of GW when co-expressed with pan-cellular mRFP1-Dyrk1b. U–X) Cytoplasmic restriction of GNESW when co-expressed with pan-cellular mRFP1-Dyrk1b.

Figure 3. Fusion of Wdr68 to the Mad repression domain is not tolerated in vivo.

A–E) Ventral views of alcian blue stained animals with the Meckel’s (M), palatoquadrate (PQ) and ceratohyal (CH) labeled in red. A) Wildtype sibling. Red arrow indicates the M-PQ jaw joint. B) Severe craniofacial defects in wdr68 ^hi3812 animals and failure of GFP mRNA to rescue. C) Rescue of the severe craniofacial defects by injection of FlagWdr68 (FW) mRNA. Red arrow indicates the mild M-PQ joint fusion phenotype. D) Severe craniofacial defects in wdr68 ^hi3812 animals and failure of MadFlagWdr68 (MFW) mRNA to rescue. 52/52 mutants genotyped were wdr68 ^hi3812 homozygotes. E) Rescue of the severe craniofacial defects by injection of CebpFlagWdr68 (CFW) mRNA. Red arrow indicates the mild M-PQ joint fusion phenotype. F) Graph summarizing the results of four injection trials. In aggregate, n = 112/452 (25%) for GFP animals, n = 16/352 (4.5%) for FW animals, n = 96/296 (32%) for MFW animals, and n = 2/149 (1.3%) for CFW animals.

Figure 4. Wdr68 is important for Myog expression in differentiating C2C12 cells.

A–D) Overlay of DAPI and Alexa-488 detection of Myog expression. A) Non-target (nt) control cells in growth medium (GM). B) nt control cells expressing Myog after 48 hours in differentiation medium (DM). C) Severely reduced expression of Myog in dyrk1b-KD cells. D) Reduced expression of Myog in wdr68-KD2 cells. E) Graph summarizing the number of Myog+ cells results for eight independent trials.

Figure 5. Wdr68 and derivatives modulate transcriptional activation of a myog promoter-reporter construct in C2C12 cells.

C2C12 cells transfected with myog-luciferase and SV40-renilla reporters. Grey columns are cells in growth medium (GM). Black columns are cells in differentiation medium (DM). The y-axis units are the same for parts A and B. A) Column pairs 1) empty pCS2+ expression vector control showing endogenous basal levels of reporter induction, 2) 100 ng MyoD further enhances reporter activity 7-fold above control. B) Column pairs 3) basal control, 4) 200 ng FlagWdr68 (FW) further enhances reporter activity 2.4-fold above control, 5) 200 ng MadFlagWdr68 (MFW) represses reporter activity to 27% of control, 6) 200 ng CebpFlagWdr68 (CFW) further enhances reporter activity 2-fold, 7) 400 ng FW further enhances reporter activity 6-fold, 8) 400 ng MFW enhances reporter activity only 2-fold, 9) 400 ng CFW further enhances reporter activity 6.4-fold.