ZFIN ID: ZDB-FIG-140522-35
Ning et al., 2013 - MicroRNA-92a upholds Bmp signaling by targeting noggin3 during pharyngeal cartilage formation. Developmental Cell   24(3):283-295 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Gene:
Antibodies:
Fish:
Condition:
Knockdown Reagent:
Anatomical Terms:
Stage Range: High-pec to Pec-fin
PHENOTYPE:
Fish:
Conditions:
Observed In:
Stage Range: High-pec to Day 4

Fig. 4 mir92a Regulates Bmp Signaling during Pharyngeal Cartilage Formation

(A–C) p-Smad1/5/8 levels were reduced in the pharyngeal region of mir92a morphants. 92a-rMO and 92a-MO-injected (4 ng) Tg(fli1:EGFP) embryos at 42 hpf were stained for p-Smad1/5/8 using anti-p-smad1/5/8 and DAPI (A, B). The pharyngeal region (A) and trunk somite muscle (B) were observed after immunostaining by confocal microscopy. p-Smad1/5/8 in cell lysis of head region at 42 hpf was detected by western blotting (C). Scale bars, 20 µm.

(D) Embryos depleted of Bmp activity showed severe pharyngeal cartilage defects. Dorsomorphin-treated wild-type (WT) or heat-shocked Tg(hsp70l:dnBmpr-GFP) transgenic live embryos were photographed at 96 hpf and Pharyngeal cartilages of heat-shocked transgenic embryos at 80 hpf were detected with Alcian blue. Scale bars, 200 µm.

(E) Embryos depleted of Bmp activity showed a reduced proliferation of pharyngeal chondrogenic progenitors. BrdU-treated Tg(fli1:EGFP) transgenic embryos were stained at 42 hpf with anti-BrdU (red) and anti-GFP antibodies (green). The pharyngeal regions were observed by confocal microscopy. Scale bar, 20 µm.

(F) Deficiency of pharyngeal chondrogenic progenitor differentiation in dorsomorphin-treated embryos. Confocal sections of the palatoquadrate (pq) and ceratohyal (ch) cartilages of Tg(fli1:EGFP) transgenic embryos. These embryos were treated with DMSO or 10 µM dorsomorphin at 36 hpf and probed with anti-GFP and anti-Col2 antibodies at 66 hpf. Scale bars, 20 µm.

(G and H) 92a-MO-induced pharyngeal cartilage defects were rescued by bmp4 overexpression. Wild-type embryos were injected with 4 ng 92a-MO alone or together with 7 pg bmp4 mRNA at the one-cell stage and stained with Alcian blue for cartilages at 80 hpf. Coinjected embryos showed different levels of recovery of neurocranium and pharyngeal cartilages. Representative cartilage phenotypes (BR0-BR3) were shown in (G) and their ratios were shown in (H).

See also Fig. S4.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP y1Tg control High-pec pharyngeal arch IHC
Pec-fin ceratohyal cartilage IHC
Pec-fin palatoquadrate cartilage IHC
y1Tg chemical treatment: pharmaceutical High-pec pharyngeal arch IHC
Pec-fin ceratohyal cartilage IHC
Pec-fin palatoquadrate cartilage IHC
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Qualifier Anatomy
Ab1-col2a IHC y1Tg control Pec-fin ceratohyal cartilage
IHC Pec-fin palatoquadrate cartilage
IHC y1Tg chemical treatment: pharmaceutical Pec-fin Not Detected ceratohyal cartilage
IHC Pec-fin Not Detected palatoquadrate cartilage
Ab1-smad IHC WT control High-pec pharyngeal arch
IHC High-pec somite
IHC WT + MO2-mir92a standard conditions High-pec pharyngeal arch
IHC High-pec somite
Phenotype Details
Fish Conditions Stage Phenotype
WT chemical treatment: pharmaceutical Day 4 head decreased size, abnormal
Day 4 pharyngeal arch absent, abnormal
w30Tg heat shock Day 4 head decreased size, abnormal
Day 4 pharyngeal arch absent, abnormal
y1Tg chemical treatment: pharmaceutical High-pec pharyngeal arch cell population proliferation decreased process quality, abnormal
Pec-fin ceratohyal cartilage chondroblast disorganized, abnormal
Pec-fin ceratohyal cartilage chondrocyte differentiation process quality, abnormal
Pec-fin palatoquadrate cartilage chondroblast disorganized, abnormal
Pec-fin palatoquadrate cartilage chondrocyte differentiation process quality, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 24(3), Ning, G., Liu, X., Dai, M., Meng, A., and Wang, Q., MicroRNA-92a upholds Bmp signaling by targeting noggin3 during pharyngeal cartilage formation, 283-295, Copyright (2013) with permission from Elsevier. Full text @ Dev. Cell