Cell Biology

BMP signaling maintains auricular chondrocyte identity and prevents microtia development by inhibiting protein kinase A

Ruichen Yang
Hongshang Chu
Hua Yue
Yuji Mishina
Zhenlin Zhang
Huijuan Liu author has email address
Baojie Li author has email address

Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
Department of Osteoporosis and Bone Diseases, Shanghai Clinical Research Center of Bone Disease, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China
Department of Biologic and Materials & Prosthodontics, University of Michigan School of Dentistry, Ann Arbor, MI, United States
Institute of Traditional Chinese Medicine and Stem Cell Research, Chengdu University of Traditional Chinese Medicine, Chengdu, China

https://doi.org/10.7554/eLife.91883.2

Open access
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Figures and data

Prrx1 marks auricle chondrocytes but few epiphyseal or articular chondrocytes.
A. Genetic tracing experiments showed that Prrx1 marks all chondrocytes in the auricle. Upper panel: a diagram showing the schedule for TAM administration and mouse euthanasia. Scale bars=50 μm.
B. Genetic tracing experiments showed that Prrx1 marks few cells in the articular cartilage or the growth plates of femurs and the growth plates of tibias in adult mice. Scale bars (femur)=100 μm. Scale bars (tibia)=50 μm.
C. Representative immunostaining results for Vimentin, αSMA, Col1α1, and Col1α2 in ear sections of Prrx1-CreERT; R26^tdTomato mice. Scale bars=50 μm.

Ablation of Bmpr1a in Prrx1⁺ cells in adult mice led to microtia.
A. A decrease in p-Smad1/5/9 on the ear sections of the Prrx1-CreERT; Bmpr1a^f/f mice. Upper panel: Schedule for TAM administration and mouse euthanasia. Scale bars=20
A. μm. Arrows: positive signals. Smad1/5/9 staining results.
B. qPCR analysis of Bmpr1a mRNA in the ear samples of the Prrx1-CreERT; Bmpr1a^f/f and control mice. N=3.
C. The ear phenotypes of the Prrx1-CreERT; Bmpr1a^f/f mice 2 months after TAM administration to adult male mice. Right panel: quantitation data. N=4.
D. H/E staining of ear sections from the mutant and control mice. Right panels: The thickness of the cartilage and the size of the chondrocytes in the ears of the mutant and control mice. Scale bars=50 μm. N=3. Unpaired two-tailed Student’s t test were applied to evaluate the correlation data in (B and C). Two-way ANOVA (or mixed model) multiple comparisons were applied to evaluate the correlation data in (D), p<0.05 was considered as statistically significant.

Ablation of Bmpr1a in Prrx1⁺ cells in young mice led to microtia.
A. A decrease in p-Smad1/5/9 on the ear sections of the Prrx1-CreERT; Bmpr1a^f/f mice that received TAM at P21. Upper panel: Schedule for TAM administration and mouse euthanasia. Scale bars=20 μm. Arrows: positive signals.
B. qPCR analysis of Bmpr1a mRNA in the ear samples of the Prrx1-CreERT; Bmpr1a^f/f and control mice. N=3.
C. The ear phenotypes of the adult Prrx1-CreERT; Bmpr1a^f/f mice that received Tam at P21. Right panel: quantitation data. N=4.
D. H/E staining of ear sections from the mutant and control mice. Right panels: The thickness of the cartilage and the size of the chondrocytes in the ears of the mutant and control mice. Scale bars=50 μm. N=3. Unpaired two-tailed Student’s t test were applied to evaluate the correlation data in (B and C). Two-way ANOVA (or mixed model) multiple comparisons were applied to evaluate the correlation data in (D), p<0.05 was considered as statistically significant.

Alteration of transcription profiles in Bmpr1a-deficient chondrocytes.
A. Heatmaps of the top 500 genes expressed in the distal part of the auricle of the Prrx1-CreERT; Bmpr1a^f/f and age- and gender-matched control mice. N=3.
B. KEGG analysis results of the pathways affected by Bmpr1a ablation.
C. GO analysis results of the modules affected by Bmpr1a ablation.

Bmpr1a deficiency leads to an auricle chondrocyte fate switch to osteoblasts.
A. Heatmaps of osteoblast-related genes and chondrocyte-related genes differentially expressed in the Bmpr1a-deficient and control mice. N=3.
B. Immunostaining results for Col1α1 in ear sections of the Bmpr1a-deficient and control mice. Scale bars=20 μm. Arrows: positive signals.
C. ALP activity was greatly increased in the auricle of the Prrx1-CreERT; Bmpr1a^f/f mice compared to the control mice. Scale bars=50 μm. Arrows: positive signals.
D. WB results showed that the expression of the osteoblast marker genes Runx2 and osteocalcin was increased in the mutant samples. Right panel: quantitation data. N=3. Unpaired two-tailed Student’s t test were applied to evaluate the correlation data in (D), p<0.05 was considered as statistically significant.

Bmpr1a deficiency led to hyperactivation of PKA signaling.
A. Immunoassaying for p-Creb in the ear sections of the Bmpr1a-deficient and control mice.
B. Western blot analysis of p-Creb in auricle samples of the Bmpr1a-deficient and control mice. Right panel: quantitation data. N=3.
C. qPCR results for Adcy5 and Adcy8 in auricle samples of the Bmpr1a-deficient and control mice. N=3. Unpaired two-tailed Student’s t test were applied to evaluate the correlation data in (B and C), p<0.05 was considered as statistically significant.

Inhibition of PKA signaling blocks microtia development in Bmpr1a-deficient mice
A. Diagram showing the schedule of TAM and H89 administration.
B. Immunostaining results of p-Creb in ear sections of the H89-treated mutant and control mice. Scale bars=50 μm.
C. The rescue of the microtia phenotype in the mutant mice by H89. Right panels: Quantitation data. N=3.
D. H/E staining results. Right panels: the thickness of the cartilage and the size of the chondrocytes in the ear of the mutant and control mice. N=3. Right panels: Quantitation data. Scale bars=50 μm. N=3.
E. Immunostaining results for Col1α1 in the ear sections of the H89-treated mutant and control mice. Scale bars=20 μm. Arrows: positive signals.
F. ALP activity was greatly suppressed by H89 in the auricle of the Prrx1-CreERT; Bmpr1a^f/f mice compared to the control mice. Scale bars=50 μm. Arrows: positive signals.
G. WB blot also showed that H89 rescued the expression of the osteoblast marker genes Runx2 and osteocalcin in the mutant mice. Right panel: quantitation data. N=3. One-way ANOVA (and nonparametric or mixed) multiple comparisons were applied to evaluate the correlation data in (C, D, and G). p<0.05 was considered as statistically significant.

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