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This work was supported by the STARS Summer research program and its wonderful board of directors, as well as the Hamon Center for Regenerative Science and Medicine. We thank the UTSW Transgenic and Histo-Pathology cores for technical assistance. I would like to thank every member of the Cleaver lab for their valuable input, for their help conducting experiments, and for teaching me so many different things. I would also like to thank Susan Moran and Michael Buntyn for their recommendation letters, which helped me enter this program, and my parents who supported me every step of the way.

12. Acknowledgments

1. Abstract

2. Questions

3. Hypothesis

4. Endocardial Cyp26B1 expression in embryonic mouse hearts

Figure 2. Cyp26B1 expression is endothelial-specific in the endocardial cushions, as well as the elongating and remodeling embryonic heart valves. (A-E) RNA in situ hybridization was performed using antisense Cyp26B1 probe on paraffin sections of embryonic hearts at E10.5, E12.5, E14.5, and E16.5. Scale = 100 𝜇m

11. Future Directions

• Further research is needed to identify the mechanisms behind Cyp26B1 regulation of heart valve development, which might be accomplished via cell lineage tracing and inhibitor treat of OFT explants.
• In depth investigation of valve remodeling is needed.
• Are there differences in the rate of cell death, a requisite component of valve remodeling (4)?
• Determine the extent to which the neural crest cell population in the heart valves is affected following loss of Cyp26B1.
• Further investigation into the ventricular septal defects is needed.

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13. References

7. Vimentin mislocalization in Cyp26B1-/- valves

• We hypothesize that because Cyp26B1 is expressed in the endocardium, it may play a role in proper heart development.

1. Chien-Jung Lin et al. (2012), Development
2. Lin et al. (2012), Development
3. Sakabe et al. (2012), Development
4. Horne et al. (2015), J. Cardiovasc. Dev. Dis.
5. MacGrogan et al. (2014), Cold Spring Harbor Perspect. Med.

Figure 1. During development, the heart valves start out as two sets of cushions. The atrioventricular canal gives rise to the AV valves and the OFT gives rise to the SL valves. Endothelial (endocardial) cells then undergo endothelial to mesenchymal transition (EMT) and invade the cardiac jelly, leading to the presence of mesenchymal cells in the developing valves. The valves then undergo elongation and remodeling before they assume their mature appearance. Cardiac neural crest cell populations (not shown) also invade the cushions and contribute to the development of some SL valve leaflets. Adapted from (1).

- What is the role of the Cyp26B1 enzyme during embryonic heart valve remodeling?

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- What is the role of the Cyp26B1 enzyme in moderating proliferation?

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- What is the mechanism through which RA and Cyp26B1 interact in the developing heart?

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5. Abnormally enlarged SL valves in Cyp26B1-/- embryonic hearts

A

E

Cyp26B1 is required to guide semilunar valve leaflet remodeling and restrict endocardial cell proliferation

Steven Lu, Caitlin M. Braitsch, Edward Daniel, and Ondine Cleaver

Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA

B

Figure 5. Intermediate filament protein vimentin is abnormally expressed throughout Cyp26B1-/- SL valve endocardium. (A-H) Immunostaining was performed using anti-vimentin antibody on paraffin sections of WT and Cyp26B1-/- hearts at E16.5 and E18.5. Arrows indicate normal vimentin+ endocardium in WT valves. Red dotted lines mark pervasive vimentin immunopositivity in Cyp26B1-deficient SL valves. Nuclei were counterstained with DAPI.

Figure 3. SL valves are significantly enlarged in Cyp26B1-/- embryonic hearts. (A-F) Hart’s elastin IHC was performed on paraffin sections of WT (n=3) and Cyp26B1-/- (n=3) embryonic heart sections at E18.5. (G) Valve widths on paraffin sections were measured perpendicular to the longitudinal axis at the widest part of the leaflet (see dotted lines in A,D). The mean widths of SL and AV valves of WT

Retinoic acid (RA), an active metabolite of Vitamin A, is vital in embryonic development although high concentrations of the compound can also act as a teratogen. Extraneous administration of RA has been linked to transposition of the great arteries and cardiac hypertrophy among other conditions, while RA deficiency is known to cause improper heart remodeling. The outflow tract (OFT), a region of the heart that in later stages of development comprises the semilunar (SL) heart valves, is particularly responsive to RA signaling. It is further accepted that the cytochrome P450 family 26 subfamily B member 1 (CYP26B1) enzyme catabolizes RA into an inactive form, suggesting that the gene is vital in limiting the effects of RA during early development. Thus far, deletion of the Cyp26B1 gene in mice have been shown to result in defects of the embryonic limbs, hindbrain, gonads, and lungs. We hypothesized that because Cyp26B1 is expressed in the endocardium, it may also play a role in proper heart development. To that end, we performed immunohistochemistry and immunostaining on heart sections to help us identify developmental defects in Cyp26B1-deficient mice. Here, we show that Cyp26B1 knockout mice exhibited multiple heart defects, namely ventricular septal defects and abnormally enlarged SL valve leaflets. The rate of cell proliferation was significantly increased in E18.5 SL valves, which suggests SL valve hyperplasia. Further research is needed to identify the mechanisms behind this process, which might be accomplished via cell lineage tracing and inhibitor treatment of OFT explants. The hope is that by better understanding the mechanisms through which Cyp26B1 and RA interact in the heart to contribute to these phenotypes, we can gain more insight on potential methods to prevent or cure congenital heart defects such as Tetralogy of Fallot and congenital pulmonary or aortic valve stenosis.

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9. Loss of Cyp26B1 leads to additional congenital heart defects

8. Aberrant proliferation in Cyp26B1-/- remodeling heart valves

Heart valve development

Figure 8. Schematic representation of RA signaling in WT and Cyp26B1-/- endocardium during cardiac development. (A) Our data suggest that in WT heart valves, RA promotion of cell proliferation is restricted by Cyp26B1 enzyme activity. (B) Without Cyp26B1, rampant RA activity promotes cell proliferation, ultimately leading to hyperplastic heart valves.

10. Model & Conclusions

Figure 7. Ventricular septal defects occur in Cyp26B1-/- mice.

(A-B’) Immunostaining using Vimentin, pHH3, and CTnT antibodies was performed on paraffin sections of WT and Cyp26B1-/- hearts at E18.5. (A) The arrowhead indicates the ventricular septum. (B,B’) Arrows indicate the ventricular septal defect (VSD). Nuclei were counterstained with DAPI.

6. Mesenchymal and extracellular matrix proteins in Cyp26B1-/- SL valves

Figure 4. Mesenchymal transcription factor Sox9 and extracellular matrix molecule Periostin are expressed in SL valve leaflets. (A-B’) Immunostaining using 𝛼SMA, Sox9, and Periostin antibodies was performed on paraffin sections of WT and Cyp26B1-/- hearts at E18.5. Nuclei were counterstained with DAPI.

RA

and Cyp26B1-/- hearts was calculated. (H-K) Immunostaining on paraffin sections was performed using alpha Smooth Muscle Actin (𝛼SMA) and PECAM1/Endomucin (PE) antibodies. Arrows indicate SL valves. Nuclei were counterstained with DAPI (blue.) Data are shown as mean ± standard error of the mean (SEM). Statistical significance was determined by Student’s t test (**, p<0.01). IHC, immunohistochemistry; ns, not significant; VSD, ventricular septal defect.

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Atrioventricular (AV) valves

Semilunar (SL) valves

LV

EMT

EMT

Figure 6. Cell proliferation was significantly increased in hyperplastic Cyp26B1-/- SL valves at E18.5. (A-H) Immunostaining was performed using vimentin, pHH3, and cTNT antibodies on paraffin sections of WT and Cyp26B1-/- hearts at E16.5 and E18.5. (I,J) The proportion of pHH3+ valve cells per section was quantified. Data are represented as mean ± SEM. Statistical significance was calculated using Student’s t test (*, p<0.05). cTnT, cardiac troponin T; pHH3, phospho-histone H3; ns, not significant

A

B

200 𝜇m

200 𝜇m

200 𝜇m

’

Elongation

Remodeling

Remodeling

Elongation