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ZFIN ID: ZDB-FIG-151214-70
Askary et al., 2015 - Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State. Developmental Cell   35:358-65 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Anatomical Terms:
Stage Range: Long-pec to Day 5
PHENOTYPE:
Fish:
Observed In:
Stage Range: Long-pec to Day 5

Fig. 3

Irx7 Represses Chondrocyte Maturation

(A and B) trps1:GFP (green) is reduced at the hyoid joint (arrows) of irx7el538 mutants compared with WT siblings. sox10:dsRed labels hyoid cartilages (red).

(C and D) Immunostaining shows increased Col2a1a protein (green) but no change in phospho-Smad1/5/8 (red) in the hyoid joints (arrows) and Hm-Sy connecting zone (arrowheads) of irx7el538; irx5ael574 mutants relative to WT siblings.

(E–H) In situ hybridization shows increased expression of col2a1a and acana in the hyoid joint region (dashed areas) of irx7el538 mutants relative to WT siblings. Numbers indicate proportion of animals showing the displayed phenotype. Scale bars represent 20 µM.

(I) Irx7 and murine IRX1 (but not GFP control) inhibit Alcian+ matrix accumulation in micromass cultures of ATDC5 cells grown in chondrogenic media for 7 days. Results were consistent across biological triplicates.

(J) Alcian blue content of the micromasses quantified by absorbance at 620 nm.

(K) qRT-PCR on RNA extracted from micromass cultures of ATDC5 cells grown in chondrogenic media for 7 days. Compared with GFP-expressing controls, expression of Col2a1, Acan, and Trps1 was reduced by IRX1 and Irx7 misexpression. Sox9 expression was reduced by IRX1 but not Irx7 misexpression. Error bars represent 95% confidence interval of the mean. p < 0.05 and p < 0.01 using Tukey HSD test.

See also Figure S3.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
acana WT standard conditions Long-pec pharyngeal arch 2 skeleton chondrocyte ISH
Protruding-mouth pharyngeal arch 2 skeleton chondrocyte ISH
irx7el538/el538 standard conditions Long-pec pharyngeal arch 2 skeleton joint ISH
Protruding-mouth pharyngeal arch 2 skeleton joint ISH
col2a1a WT standard conditions Long-pec pharyngeal arch 2 skeleton chondrocyte ISH
Protruding-mouth pharyngeal arch 2 skeleton chondrocyte ISH
irx7el538/el538 standard conditions Long-pec pharyngeal arch 2 skeleton joint ISH
Protruding-mouth pharyngeal arch 2 skeleton joint ISH
DsRed el10Tg ; trps1j1271aGt standard conditions Day 5 pharyngeal arch 2 skeleton chondrocyte IFL
irx7el538/el538 ; trps1j1271aGt ; el10Tg standard conditions Day 5 pharyngeal arch 2 skeleton chondrocyte IFL
EGFP el10Tg ; trps1j1271aGt standard conditions Day 5 pharyngeal arch 2 skeleton joint IFL
irx7el538/el538 ; trps1j1271aGt ; el10Tg standard conditions Day 5 pharyngeal arch 2 skeleton joint IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-col2a IHC WT standard conditions Protruding-mouth pharyngeal arch 2 skeleton chondrocyte
IHC irx5ael574/el574 ; irx7el538/el538 standard conditions Protruding-mouth pharyngeal arch 2 skeleton chondrocyte
IHC Protruding-mouth pharyngeal arch 2 skeleton joint
Ab1-smad IHC WT standard conditions Protruding-mouth pharyngeal arch 2 skeleton chondrocyte
IHC irx5ael574/el574 ; irx7el538/el538 standard conditions Protruding-mouth pharyngeal arch 2 skeleton chondrocyte
Phenotype Details
Fish Conditions Stage Phenotype
irx5ael574/el574 ; irx7el538/el538 standard conditions Protruding-mouth ceratohyal-interhyal joint chondrocyte development increased process quality, abnormal
Protruding-mouth interhyal-hyosymplectic joint chondrocyte development increased process quality, abnormal
Protruding-mouth pharyngeal arch 2 skeleton embryonic skeletal joint development decreased process quality, abnormal
Protruding-mouth pharyngeal arch 2 skeleton joint ab1-col2a labeling increased amount, abnormal
irx7el538/el538 standard conditions Long-pec pharyngeal arch 2 skeleton embryonic skeletal joint development decreased process quality, abnormal
Long-pec pharyngeal arch 2 skeleton joint acana expression increased amount, abnormal
Long-pec pharyngeal arch 2 skeleton joint col2a1a expression increased amount, abnormal
Protruding-mouth ceratohyal-interhyal joint cartilage development increased process quality, abnormal
Protruding-mouth interhyal-hyosymplectic joint cartilage development increased process quality, abnormal
Protruding-mouth pharyngeal arch 2 skeleton embryonic skeletal joint development decreased process quality, abnormal
Protruding-mouth pharyngeal arch 2 skeleton joint col2a1a expression increased amount, abnormal
Protruding-mouth pharyngeal arch 2 skeleton joint acana expression increased amount, abnormal
irx7el538/el538 ; trps1j1271aGt ; el10Tg standard conditions Day 5 pharyngeal arch 2 skeleton embryonic skeletal joint development decreased process quality, abnormal
Day 5 pharyngeal arch 2 skeleton joint EGFP expression decreased amount, 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, 35, Askary, A., Mork, L., Paul, S., He, X., Izuhara, A.K., Gopalakrishnan, S., Ichida, J.K., McMahon, A.P., Dabizljevic, S., Dale, R., Mariani, F.V., Crump, J.G., Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State, 358-65, Copyright (2015) with permission from Elsevier. Full text @ Dev. Cell