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First published online 8 April 2004
doi: 10.1242/dev.01094

Development 131, 2219-2231 (2004)
Published by The Company of Biologists 2004
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BMP10 is essential for maintaining cardiac growth during murine cardiogenesis

Hanying Chen1, Shu Shi1, Lourdes Acosta2, Weiming Li1, Jonathan Lu3, Shideng Bao4, Zhuang Chen1, Zuocheng Yang1, Michael D. Schneider5, Kenneth R. Chien3, Simon J. Conway1, Mervin C. Yoder1, Laura S. Haneline1, Diego Franco2 and Weinian Shou1,*

1 Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA
2 Department of Experimental Biology, University of Jaen, Jaen 23071, Spain
3 Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA
4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27708, USA
5 Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA



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  		Fig. 3. Morphological and histological analysis of BMP10-deficient embryos and hearts. (A) (a,b,e,f,k,l,q,r) comparison of gross morphology of normal littermate control and BMP10-deficient embryos from E8.75 to E10.5. (a,b) No apparent abnormality was detected at E8.75. (e,f) Some BMP10-deficient embryos were slightly growth retarded at E9.0. (k,l) Severe growth retardation was seen in BMP10-deficient embryos at E9.5; however, mutants had an identical number of somite pairs and normal allantoic connection when compared with littermate controls. Over 50% of BMP10-deficient embryos had severe edema and expanded pericardiac sacs, suggesting poor cardiac function in these mutants. (q,r) BMP10-deficient embryos were dead by E10.5. (c,d,g,h,I,j,m,n,o,p,s,t) comparison of histological sections of normal control and BMP10-deficient hearts from E8.75 to E10.5 embryos stained with haematoxylin and eosin. (d,h,j) At E8.75-E9.0, BMP10-deficient embryos had normal rightward looped heart and primitive ventricular chambers, suggesting that BMP10 is not required for the early phases of cardiogenesis. Also, the size of the heart in BMP10-deficient embryos was grossly normal compared with littermate control but exhibited some thinned myocardium (white arrow). Acellular endocardial cushions were formed in both the outflow track (OFT) and atrial-ventricular canal (AVC). (n,p,t) Compared with wild-type normal hearts at E9.5-E10.5, BMP10-deficient hearts were growth retarded, had hypoplastic walls and failed to develop normal ventricular trabeculae and endocardial cushions. While endocardial cushions in OFT and AVC of wild-type control hearts had begun to be seeded after epithelial-mesenchymal transformation of adjacent endocardium (black asterisks in o and s), acellular endocardial cushions remained in BMP10-deficient hearts (white asterisks). (B) Ink injection was used to visualize the cardiac contractile function and blood flow in E9.0 and E9.5-E9.75 embryos. (a,c) Ink injected in the primitive left ventricle was efficiently pumped throughout the entire cardiovascular system in control embryos. (b) At E9.0, the circulation was established in BMP10-deficient embryos, however, not as efficiently as littermate controls, which might reflect the weaker/slower heart rate in BMP10 mutants. (d) At E9.5-9.75, ink remained in the BMP10-deficient ventricles, suggesting poor cardiac function. Note that the ink within the BMP10-deficient heart has diffused in a retrograde direction into the sinus venous (yellow arrow) and yolk sac (green arrow) due to lack of adequate cardiac contraction and circulation. Red arrows indicate circulated ink around the embryonic head region. Blue arrows indicate hearts. The posterior portions of the embryos were removed to visualize the hearts (c,d).

 


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  		Fig. 1. Expression pattern of BMP10 during cardiogenesis. (A) In (a-d), using whole-mount in-situ hybridization, BMP10 transcripts were not detected in E8.5 embryonic hearts (a), but were detected in developing ventricles and atria at E9.5 (b), and at E11.5 (c). BMP10 expression was restricted to trabecular myocardium. Red arrows indicate the heart in (a,b) and trabecular myocardium in (c), and blue arrows indicate the ventricular compact wall. (d) BMP10 expression was detected only in atria at E18.5. In (e-i), in-situ hybridizations were performed on sagittal sections of E11.5, transverse sections of E13.5 embryos, and transverse sections of an adult mouse heart. (e) Myosin heavy chain ß (MHCß) transcripts were detected throughout the myocardium. (f) A BMP10 sense probe was used as a negative control. (g,h) BMP10 expression in the ventricle was restricted to trabecular myocardium and (i) became restricted to the right atrium in adults. (j) BMP10 transcripts were detected in the heart as early as E8.75 using RT-PCR. By E16.5, BMP10 was hardly detectable in ventricles. (k) Using northern blot to confirm BMP10 expression in FKBP12-deficient hearts (E14.5). BMP10 transcripts were significantly upregulated in FKBP12-deficient hearts. (B) Multiple-tissue quantitative mRNA level analysis. (a) Quantitative dot blot analysis of evenly loaded 84 mRNA samples from varying adult human tissues (Clontech). Only mRNA samples isolated from whole heart (A4) and right atrium (D4) showed significant BMP10 mRNA level when compared to negative control samples. (b) Phosphorimage analysis on the relative level of expression of BMP10 in different regions of the adult heart. The phosphorimaging values were corrected for the mean background, consisting of negative controls (yeast total RNA, yeast tRNA, E. coli rRNA, E. coli DNA) and empty fields.

 


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  		Fig. 2. Generation of BMP10-deficient mice. Targeting vector to mutate the mouse BMP10 gene in embryonic stem cells (a), Southern blot analysis of genomic DNA (digested with EcoRV and probed with 3'-probe) derived from a single litter of E9.5 embryos after mating of bmp10m1/+ mice (b), and RT-PCR analysis to confirm the inactivation of BMP10 expression in bmp10m1/bmp10m1 embryos (E9.0) (c). Expression of FKBP12 was used as a loading control.

 


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  		Fig. 4. Whole-mount immunostaining and confocal microscopic analysis of control (A,C) and BMP10-deficient embryos (B,D) at E9.25. Fluorescence conjugated anti-Flk-1 monoclonal antibody stains the endothelial cells (green) in the developing vasculature and endocardium of the developing heart, while anti-myosin heavy chain monoclonal antibody MF-20 stains the myocardium (blue). (A,B) comparison of endothelial development in both control and BMP10-deficient embryos at E9.25. Endothelial development was not affected in BMP10-deficient embryos. (C,D) Comparison of endocardium and myocardium development in control and BMP10-deficient ventricles. The BMP10-deficient heart displayed a much thinner ventricular wall compared with the control heart; however, the endocardium was in normal proximity to the myocardium. Some primitive trabeculae were formed in the BMP10-deficient ventricles at this age. Red arrows point to the primitive trabecular myocardium, while white arrows indicate the endocardium.

 


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  		Fig. 5. Distribution and expression of p57kip2 in BMP10-deficient and FKBP12-deficient hearts using immunohistochemistry staining. (A) At E9.5, p57kip2 expression was undetectable in the wild-type heart (a) but was ectopically present in the BMP10-deficient heart (b,c) under identical staining conditions. The expression of p57kip2 was abundant in E13.5 ventricular trabecular myocardium of the wild-type heart (d) but was significantly downregulated in the E13.5 FKBP12-deficient trabecular myocardium (e). Arrows indicate areas of positive staining. (B) Using RT-PCR to confirm the mRNA level of p57kip2 in BMP10-deficient hearts (E9.5) and FKBP12-deficient hearts (E13.5). The expression of p57kip2 was upregulated in BMP10-deficient hearts and downregulated in FKBP12-deficient hearts.

 


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  		Fig. 6. Analysis of cardiac markers in BMP10-deficient hearts. Using in-situ hybridization to analyze the expression of cardiac markers at E9.5. The cardiac chamber markers MLC2v (A,B) and MLC2a (C,D) were not altered in the BMP10-deficient hearts, suggesting that cardiac patterning and chamber specification are normal in the BMP10-deficient heart. Cardiogenic transcription factors NKX2.5 (E,F) and MEF2C (G,H) were significantly downregulated at E9.5 in the BMP10-deficient hearts. The expression of Chisel (I,J) and ANF (K,L) was also reduced in the BMP10-deficient heart, while the expression of HOP (M,N) remained at a similar level in the BMP10-deficient heart compared with the control heart. a, atrium; v, ventricle; of, outflow tract.

 


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  		Fig. 7. The effect of BMP10 on cultured cardiomyocytes and hearts. (A) Generation of BMP10-expressing NIH 3T3 cell lines and using cardiomyocyte co-culture assay to determine the biological activity of BMP10. (a) Schematic diagram of BMP10 retroviral vector used to express BMP10. (b) Co-culture of embryonic cardiomyocytes with BMP10 feeder cells (NIH3T3/BMP10) and control feeder cells (NIH3T3/EGFP). The proliferative activity of cardiomyocytes in culture after 24, 48 and 72 hours was determined using [3H]thymidine labeling index of PAS positive cells as described in Materials and methods. BMP10 feeders were able to maintain higher proliferative activity of cardiomyocytes compared with control feeder cells. (B) In-vitro culture of embryonic hearts in BMP10-conditioned and control media. Embryonic hearts were isolated from E9.0-E9.25 embryos harvested from bmp10m1/+ matings. Each heart was photographed before (a) and after (b) 24 hours of culture. (c) Hematoxylin- and eosin-stained histological sections of cultured hearts. (d) [3H]thymidine labeling was used to determine the proliferative activity of cultured hearts. Autoradiographs of [3H]thymidine labeled hearts. (e) Hoechst staining to show nuclei. The images of each column were from the same heart. (C) [3H]thymidine labeling index of cultured embryonic hearts. (D) Heart rates of BMP10-deficient and control hearts prior to culture, and following culture in control and BMP10-conditioned media. BMP10-conditioned media was able to rescue the heart rates of BMP10-deficient embryos.

 


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  		Fig. 8. BMP10-conditioned medium restored the normal genetic program in BMP10-deficient hearts. (A) Whole-mount in-situ hybridization of NKX2.5 expression in cultured embryonic hearts. Embryonic hearts were isolated at E9.0 and cultured in BMP10-conditioned media overnight. Prior to the culture, the BMP10-deficient hearts (n=3) (a) had a significantly lower NKX2.5 expression than the littermate control hearts (n=3) (b). After culture, the BMP10-deficient hearts (n=4) (c) had restored NKX2.5 expression. (d) Littermate control heart (n=3) cultured in BMP10-conditioned medium. (B) Immunohistological staining of p57kip2 expression in cultured embryonic hearts. Embryonic hearts were isolated at E9.0 and cultured in BMP10-conditioned media or control medium overnight. (a) Wild-type hearts cultured in control medium (n=6). (b) BMP10-deficient hearts cultured in control medium (n=4). (c) BMP10-deficient hearts cultured in BMP10-conditioned medium (n=4). The expression of p57kip2 in mutant hearts cultured in BMP10-conditioned medium was significantly downregulated when compared with the mutant hearts cultured in control medium.

 


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  		Fig. 9. A model for the modulation of cardiac growth and function by BMP10 during mid-gestation. FKBP12 negatively regulates BMP10 possibly via its interaction to type I receptor for BMP10. BMP10 has double biological activities: (1) it prevents the premature activation and/or antagonizes the activity of negative cell cycle regulators such as p57kip2; (2) it maintains cardiac function by regulating the level of expression of several key cardiogenic transcriptional factors during mid-gestation.

 

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© The Company of Biologists Ltd 2004