Poster produced by Faculty & Curriculum Support (FACS), Georgetown University School of Medicine

Detailed analysis of the morphology of mouse embryos with a point mutation in non-muscle myosin heavy chain II-B using high resolution 3D imaging

Zachary M. Weisner1,2; Xuefei Ma Ph.D.2; and Robert S. Adelstein M.D.2

Abstract

1 Georgetown University Medical Center, Department of Biochemistry and Molecular Biology, Washington, DC2 National Institutes of Health, National Heart, Lung, and Blood Institute, Laboratory of Molecular Cardiology,

Bethesda, Maryland

Introduction

Methods & Materials

Conclusions

Future Directions

References

Acknowledgments

Mutations of the Myh10 gene, encoding nonmuscle myosin heavy chain II-B (NMHC II-B), are proposed to be one of the causative genetic links to Pentaology of Cantrell (POC). POC is a developmental defect causing inadequate closure of the ventral body wall often resulting in ectopia cordis and an omphalocele in 63% of all cases. POC presents with a set of five phenotypes including: (1) anterior diaphragm deficiency, (2) midline supraumbilical wall defect, (3) diaphragmatic pericardium defect, (4) intracardiac abnormalities such as double outlet right ventricle (DORV) and ventral septal defect (VSD), and (5) lower sternum defect (Ma and Adelstein). POC is a very rare congenital defect that affects up to 1 in 200,000 live births (Harmath, Hajdu and Hauzman). Males are generally affected more than females with a predominance of 2.7:1 (Pakdaman, Woodward and Kennedy). In efforts to better understand the phenotypic development over time, our lab utilized a mouse model with a point mutation at R709C on NMHC II-B. This study performed whole body micro-computerized topography scans on mice at embryonic day (E)14.5, allowing characterization of the defects. Hearts were then dissected and a detailed CT scan was performed. Preliminary results reveal that mice homozygous for the R709C mutation contain all the fore- mentioned defects. Heterozygous mice for the R709C mutation vary in their phenotype expression.

Review of Nonmuscle Myosin II-B:

I would like to sincerely thank Dr. Xuefei Ma and Dr. Robert S. Adelstein for their guidance and instruction throughout this project. A special thanks to Dr. Mary Anne Conti for a review of this poster. Additionally, I would like to express gratitude to Danielle Donahue of the Mouse Imaging Facility for her guidance with the CT programing. Finally, I would like to thank Milton Williams and Mohamed Elshazzly for their support.

Figure 1: Non-muscle myosin II-B

Genotyping: Confirming Mutant Mice Figure 5: Results of Genotyping

Figure 5 displays the genotyping results conducted for the E14.5 embryos. Embryos included MA-134, 1-8, for each respective lane. Lanes 1 and 2 were mutant, Lane 3 was wild type, and Lanes 4 thru 8 were heterozygous.

• NM-II is hypothesized to be a genetic link to POC

• Homozygous NM II-B R709C mice exhibit a phenotype consistent with all aspects of POC

• Homozygous mutation is embryonic lethal for mice at E14.5 due to cardiac failure

• Heterozygous mice vary in their phenotype expression. Some mice do not display defects suggesting other compensatory mechanisms.

• Most common heart defects include double outlet right ventricle (DORV) and ventral septal defects (VSD).

• Some studies suggest that the cardiac defects occur in order to promote survival in utero in efforts to adapt to the disease.

Figure 6: Microscope Evaluation of Embryos:

Figure 6: All the above specimens are at E14.5. 1 and 2 are mutant, 3 is a wild type and the rest are heterozygous. Current studies show that a mutation in NM II-B is embryonic lethal by E14.5.

Understanding Pentalogy of Cantrell:

Identifying the Time Line of Defects: Mouse Model

Figure 2: POC Clinical Development

Figure 2: On the left is a normal heart. In the middle, the visual defects of POC are displayed. Ectopia cordis is present. An omphalocele is present in 63% of all cases. The five clinical phenotypes include: (1) anterior diaphragm deficiency, (2) midline supraumbilical wall defect, (3) diaphragmatic pericardium defect, (4) intracardiac abnormalities such as double outlet right ventricle (DORV) and ventral septal defect (VSD), and (5) lower sternum defect. On the right are cartoon displays of the DORV and VSD defects associated with the heart.

Results

Results

Figure 1: There are three isoforms of nonmuscle myosin II (NM II), including: A, B, and C. Myosin interacts with actin in order to facilitate cell division, cell-cell adhesion, and cell migration. The N-terminal makes up the globular domain and the C-terminal makes up the coiled-coil end of the myosin heavy chain. Two sets of light chains bind near the globular domain. These two pairs of light chains are essential for function and regulation. Myh10 is the gene encoding NMHC II-B. NM II-B is shown to be essential for cardiac development. Mutations in the Myh10 gene are hypothesized to be related to POC.

Figure 3: Mouse Embryonic Development Diagram

Figure 4: Cardiac Development in Mouse Embryos

Figure 3: The embryonic development of mice occurs within 18-21 days of gestation. A zygote undergoes gastrulation and turning and eventually organogenesis followed by birth. Lifespan averages about 867 days.

Figure 4: The cardiac development of the heart starts around E7.5 with the formation of the cardiac crescent. The heart tube and looping heart form by E8.5. The chamber formation is defined by E10.5 and the tissues become defined by E15. NM II-B Mice which phenocopy POC, die at E14.5 due to cardiac failure.

Micro-Computerized Topography: Figure 7: Micro-CT Scans with a sagittal plane view:

Histology and Immunohistochemistry: Figure 8: H&E staining on Cardiac Structures

Figure 9: Immunofluorescence Confocal Microscopy

Figure 8 displays cardiac histology of a wild type E14.5 embryo. On the left side is the 10x view of the heart and on the left is a 40x view, focused on the cardiac myocytes of the myocardium.

Figure 7: A full body CT was performed on the fore-mentioned samples. Three fields of view were included. Red arrows indicate the heart.

Figure 9: E14.5 mouse sagittal view of the heart. Top row, NM II-A (green), E-cadherin (red), and DAPI stains nuclei (blue). Bottom row, NM II-B (green), desmin (red), and DAPI stains nuclei (blue). Images were obtained with confocal microscopy. Z stacks were obtained with heart tissue and cells. The cell images were taken at 63x and the tissues were viewed at 20x both with confocal microscopy.

• Complete micro-CT scanning for mouse embryos. Our group plans to analyze mice from E10 to E14. The goal is to determine a time line of defects.

• Continue heart dissection of the embryos and perform detailed scans of the heart to better characterize the cardiac defects.

• Determine the mechanism behind POC and any related cellular pathways.

• Determine other causative genetic links with POC.

1. Alghamdi, A. A., & Odim, J. (2015, January 30). Double Outlet Right Ventricle Surgery. Retrieved from Medscape Refrence: http://emedicine.medscape.com/article/904397-overview#a04

2. Harmath, Agnes, et al. "Surgical and Dysmorphological Aspects of Abdominal Wall Defects." Donald School Journal of Ultrasound in Obstetrics and Gynecology (2009): 38-47. Document.

3. Kim, A. J., Francis, R., Liu, X., Devine, W. A., Ramirez, R., Anderton, S. J., . . . Lo, C. W. (2013, July). Micro-computed Tomography Provides High Accuaracy Congenital Heart Disease Diagnosis in Neonatal and Fetal Mice. Circ Cardiovasc Imaging, 6(4), 551-559.

4. Liu, X., Tobita, K., Francis, R. J., & Lo, C. W. (2013, June ). Imaging Techniques for Visualizing and Phenotyping Conenital Heart Defects in Murine Models. Birth Defects Res C Embryo Today, 93-105.

5. Ma, X., & Adelstein, R. S. (2014, May/June). The role of vertebrate nonmuscle Myosin II in development and human disease. BioArchitecture, 4(3), 88-102. Retrieved 11 2014

6. Pakdaman, R., Woodward, P., & Kennedy, A. (2015). Complex Abdominal Wall Defects: Appearances at Prenatal Imaging. Radio Graphics, 35, 636-649.

7. Vicente-Manzanares, M., Ma, X., Adelstein, R. S., & Horwitz, A. R. (2009, November). Non-muscle myosin II takes centre stage in cell adhesion and migration. Nature Review Molecular Cell Biology, 10, 778-790.

Morphological Characterization:

Mouse Model: A mixture of SV129 and C57BL/6J mouse models contained a mutation in the Myh10 gene. Point mutation in R709C of NM II-B.Genotyping: Samples were obtained from embryos and lysed. PCR was performed. Samples were run on a 1.0% gel and photos were taken on a UV-transilluminator.Micro-Computerized Topography: Embryos were incubated in contrast solution (25% lugol and 5% iodine) for 48 hours. Samples then were placed in a Sky Scan 1172. Reconstruction, analysis, and histogram was adjusted using Sky Scan programs. Resolution went as low as 13 microns for full body scans and 4 microns for detailed heart scans.Histology and Immunofluroescence: Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (preformed previously by XM). Samples were dewaxed and antigen retrieval was performed. Blocking buffer was 1% BSA/10% normal goat serum in PBS. Samples were blocked for 1 hour and primary antibody was incubated at 4˚C overnight. Secondary Ab was incubated for an hour at room temperature. Primary Abs: NM II-A with E-cadherin, and NM II-B with desmin. Images were taken on Zeiss 780 inverted confocal microscope.

AoSVC

LV RA

Harmath, Agnes, et al. "Surgical and Dysmorphological Aspects of Abdominal Wall Defects." Donald School Journal of Ultrasound in Obstetrics and Gynecology (2009): 38-47. Document.Harmath, Agnes, et al. "Surgical and Dysmorphological Aspects of Abdominal Wall Defects." Donald School Journal of Ultrasound in Obstetrics and Gynecology (2009): 38-47. Document.

DORV

VSD