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Fig. 3

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ZDB-FIG-151214-70

Publication

Askary et al., 2015 - Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State

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Fig. 3

Irx7 Represses Chondrocyte Maturation

(A and B) trps1:GFP (green) is reduced at the hyoid joint (arrows) of irx7^el538 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 irx7^el538; irx5a^el574 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 irx7^el538 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.

Expression Data

Genes:	acana col2a1a DsRed EGFP
Antibodies:	Ab1-col2a Ab1-smad
Fish:	WT irx7^el538/el538 trps1^j1271aGt; el10Tg irx5a^el574/el574; irx7^el538/el538 irx7^el538/el538; trps1^j1271aGt; el10Tg
Anatomical Terms:	pharyngeal arch 2 skeleton chondrocyte pharyngeal arch 2 skeleton joint
Stage Range:	Long-pec to Day 5

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	irx7^el538/el538 irx5a^el574/el574; irx7^el538/el538 irx7^el538/el538; trps1^j1271aGt; el10Tg
Observed In:	ceratohyal-interhyal joint cartilage development ceratohyal-interhyal joint chondrocyte development interhyal-hyosymplectic joint cartilage development interhyal-hyosymplectic joint chondrocyte development pharyngeal arch 2 skeleton embryonic skeletal joint development pharyngeal arch 2 skeleton joint
Stage Range:	Long-pec to Day 5

Phenotype Detail

Acknowledgments

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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