Atypical Protein Kinase C Dependent Polarized Cell Division Is Required for Myocardial Trabeculation

University of Nebraska Medical Center DigitalCommons@UNMC

Theses & Dissertations Graduate Studies

Fall 12-18-2015

Atypical Protein Kinase C Dependent Polarized Cell Division is Required for Myocardial Trabeculation

Derek L. Passer University of Nebraska Medical Center

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Atypical Protein Kinase C Dependent Polarized Cell Division is Required for Myocardial Trabeculation

Derek Passer

A DISSERTATION

Presented to the Faculty of

The Graduate College in the University of Nebraska

In Partial Fulfillment of the Requirements

For the Degree of Doctor of Philosophy

Department of Cellular and Integrative Physiology

Under the Supervision of Ibrahim J. Domian and Irving H. Zucker

University of Nebraska Medical Center

Omaha, Nebraska

2015

TABLE OF CONTENTS LIST OF FIGURES AND TABLES...... 4 LIST OF ABBREVIATIONS...... 6 CHAPTER I: INTRODUCTION ...... 10 Development of the Early Mouse Heart ...... 11 Cell Polarity During Embryogenesis ...... 14 The Par Polarity Complex ...... 15 Upstream Polarity Signaling Cues ...... 17 Function of the Par Complex in Multicellular Organisms ...... 17 Symmetric Polarized Divisions vs. Asymmetric Polarized Divisions ...... 18 The Protein Kinase C Family ...... 21 Essential Role of Prkci During Embryogenesis ...... 21 Hypotheses: ...... 23 CHAPTER II: MATERIALS AND METHODS ...... 25 Immunohistochemistry ...... 26 Animal breeding ...... 26 Image analysis ...... 27 Electron microscopy...... 27 Co-immunoprecipitation ...... 28 qPCR ...... 28 Ex vivo scratch assay ...... 30 Measurements, quantification, and statistical analysis ...... 30 CHAPTER III: RESULTS...... 32 Par Complex components localize asymmetrically in luminal myocardial cells...... 33 Prkci is required for polarized cell division, ventricular trabeculations, and embryonic viability...... 51 NuMA is required for polarized cell division, ventricular trabeculations, and embryonic viability...... 66 Lineage tracing of ventricular trabeculations ...... 76 CHAPTER IV: DISCUSSION ...... 81 Summary and Main Experimental Findings ...... 82 Polarity during embryogenesis ...... 82 The Heart Polarity Model ...... 83 Cardiac Jelly and Myocardial Polarity ...... 83 3

Hyaluronic acid does not signal myocardial polarity through CD44 ...... 84 The role of Prkci during early cardiogeneis ...... 85 In vitro cardiomyocyte polarization ...... 87 The role of NuMA during early cardiogeneis ...... 88 Lineage tracing of early trabeculations ...... 89 Conclusions and perspectives ...... 90 Polarity signaling during human disease ...... 90 Understanding myocardial polarity is essential for cardiovascular regenerative medicine ...... 91 CHAPTER V: REFERENCES ...... 92

LIST OF FIGURES AND TABLES Figure 1: Formation of trabeculations from the luminal surface of the primitive heart tube...... 12 Figure 2: Diagram of Early Heart Development from E7.5-E10.5...... 13 Figure 3: Partitioning (Par) complex proteins...... 16 Figure 4: Symmetric vs. Asymmetric cell Division in Epithelial Cells...... 20 Figure 5: Hypothetical Representation of the Spatial Organization of the Early Embryonic Heart...... 24 Figure 6: Luminal localization of Par complex members throughout the early embryonic heart...... 34 Figure 7: Luminal co-localization of Pard6g and Prkci in early embryonic hearts...... 35 Figure 8: Luminal localization of Par complex members in early embryonic hearts. .. 37 Figure 9: Confocal microscopy images of co-localization of Par complex members. . 38 Figure 10: Polarized cell division in early mitotic luminal myocardial cells of E8.5 hearts...... 40 Figure 11: Polarized cell division in late mitotic luminal myocardial cells of E8.5 hearts...... 42 Hyaluronic Acid (HA) in the cardiac jelly is required for Par complex localization...... 43 Figure 12: HA in the cardiac jelly is required for Par complex localization in E8.5 mouse hearts...... 44 Figure 13: HA in the cardiac jelly is required for oriented cell division in early mitotic E8.5 cardiomyocytes...... 45 Figure 14: HA in the cardiac jelly is required for oriented cell division in early mitotic E8.5 cardiomyocytes...... 46 Figure 15: Figure 3: CD44 is not required for polarized cell division in E8.5 mouse hearts...... 48 Figure 16: CD44 is not required for oriented cell division in early mitotic E8.5 cardiomyocytes...... 49 Figure 17: CD44 is not required for oriented cell division in late mitotic E8.5 cardiomyocytes...... 50 Figure 18: Genotypes of pups born from an Nkx 2.5+/cre/Prkci+/fl male crossed with a Prkcifl/fl female...... 52 Figure 19: Gross morphological analysis of an E10.5 Prkci cardiac null mouse heart. 53 Figure 20: Prkci is required for normal ventricular trabeculation...... 54 Figure 21: Comparison of endocardium from WT and Prkci cardiac null E8.5 embryos...... 55 5

Figure 22: Scanning electron microscopy and immunohistochemistry of the E10.5 Prkci conditional null heart...... 57 Figure23: RT-PCR analysis in E10.5 WT and Prkci mutant hearts...... 58 Figure 24: Localization of Par complex members in Prkci cardiac null...... 60 Figure 25: Rosa mT/mG crossed with Nkx2.5+/cre and PRKCi fl/fl ...... 61 Figure 26: Prkci is required for oriented cell division in early mitotic E8.5 cardiomyocytes...... 63 Figure 27: Prkci is required for oriented cell division in late mitotic E8.5 cardiomyocytes...... 64 Figure 28: Atypical protein kinase C activity is required to induce polarization of embryonic cardiomyocytes cultured in vitro...... 65 Figure 29: Genotypes of pups born from an Nkx 2.5+/cre/NuMA+/fl male crossed with a NuMAfl/fl female...... 67 Figure 30: Gross morphological analysis of an E10.5 NuMA cardiac null mouse heart...... 68 Figure 31: NuMA is required for normal ventricular trabeculation...... 69 Figure 32: Comparison of endocardium from WT and NuMA cardiac null E8.5 embryos...... 70 Figure 33: NuMA is required for cell polarization and normal ventricular trabeculation...... 72 Figure 34: HAS2, Prkci, or NuMA nulls do not alter cell prolfieration rates in E8.5 embryos...... 73 Figure 35: NuMA is required for oriented cell division in early mitotic E8.5 cardiomyocytes...... 74 Figure 36: NuMA is required for oriented cell division in late mitotic E8.5 cardiomyocytes...... 75 Figure 37: Polarized cell division and perpendicular spindle orientation direct trabecular formation...... 77 Figure 38: Confocal microscopy of an E9.5 Nkx 2.5+/cre R26R+/Confetti transgenic heart...... 78 Table 1: Chi-square test values for spindle orientation and cell division plane in WT vs. KO embryos...... 79 Table 2: Chi-square test values for spindle orientation and cell division plane in early mitotic vs. late mitotic myocardial cells...... 80

LIST OF ABBREVIATIONS aPKC Atypical protein kinase C cKO Conditional knockout

ECM Extracellular matrix

EM Electron microscopy

Gαi G protein ialpha subunit

HA Hyaluronic acid

HAS2 Hyaluronan synthase 2

Insc Inscuteable

LM Littermate

NuMA Nuclear mitotic apparatus protein

Par Partitioning-defective

Par3 Partitioning-defective protein 3

Par6 Partitioning-defective protein 6

Pcnt Pericentrin pHH3 Phospho-histone H3

Pins Partners of Inscuteable

Prkci Protein kinase C iota

SM Somite matched

TnT Troponin T

Atypical Protein Kinase C Dependent Polarized Cell Division is Required for Myocardial Trabeculation

Derek Passer

University of Nebraska Medical Center, 2015

Advisors: Ibrahim J. Domian M.D. Ph.D. & Irving H. Zucker Ph.D.

A hallmark of cardiac development is the formation of myocardial trabeculations exclusively from the luminal surface of the primitive heart tube. Although a number of genetic defects in the endocardium (Grego-Bessa et al., 2007; Liu et al., 2010) and cardiac jelly (Camenisch et al., 2000) disrupt myocardial trabeculation, the role of cell polarity machinery in driving this process remains unclear.

Throughout evolution, from bacteria to worms to mammals, cell polarity plays a central role in tissue development and function. How individual cells are organized and how this organization leads to the assembly of complex organs is a fundamentally important and deeply intuitive question. We hypothesized that cell polarity is an essential component to the normal development of the mammalian heart during early cardiogenesis.

Herein, we demonstrate that atypical protein kinase C iota (Prkci) and its other six interacting partners of Par polarity complex are localized to the luminal side of 8 myocardial cells on embryonic day (E) 8.5 prior to the onset of trabeculations.

Remarkably, a subset of these cells undergo polarized cell division with the mitotic spindle and the cell division plane oriented perpendicular to the heart’s lumen. Using mouse knockout models we disrupted this pathway at three separate points in the polarity pathway. First we discovered that a normal composition of the cardiac jelly is required to localize the Par polarity machinery up stream of the cardiomyocyte polarization in vivo. Disruption of the normal composition of the cardiac jelly by deletion of hyaluronic acid synthase 2 results in loss of Par complex localization, loss of polarized cell division, and loss of myocardial trabeculation.

Next we disrupted the Par polarity complex at two separate downstream points in the E8.5 mouse embryo through cardiac specific deletion of Prkci or its downstream interacting partner NuMA. We find that both deletions result in aberrant mitotic spindle alignment, loss of polarized cardiomyocyte division, and loss of normal myocardial trabeculation. These findings are further corroborated by multi-color lineage tracing experiments with state-of-the-art microscopy showing that individual trabeculations arise from a single parent cell.

Finally, using a scratch/wound assay in vitro, we show that the Golgi complex

E12.5 myocardial cells can be induced to spatially polarize towards the leading edge of the scratch. Inhibition of Prkci through a small molecule inhibitor of the protein leads to a random orientation of the Golgi complex. 9

Collectively these results layout a new mechanism for cardiac morphogenesis where in response to inductive signals from the cardiac jelly, Prkci and its downstream partners orients myocardial cells relative to the inside lumen and further directs polarized cell division of luminal myocardial cells to drive trabeculation in the early developing heart. 10

CHAPTER I: INTRODUCTION

Development of the Early Mouse Heart

During embryogenesis, cardiovascular cell types arise from multipotent progenitors in the first heart field (FHF) and in the second heart field (SHF), which are a closely associated set of cells in the developing mesoderm. The anterior lateral mesoderm forms the FHF, which gives rise to the cardiac crescent in early embryogenesis. Progenitors of the cardiac crescent then coalesce at the ventral midline to form the linear heart tube. At this point in development, the nascent heart is constituted of an inner endocardial cellular layer separated from an outer myocardial cellular layer by a complex of extracellular matrix (ECM) proteins termed the cardiac jelly (Moorman and Christoffels, 2003).

By the end of cardiac looping on embryonic day (E) 9.0-9.5 in the mouse, myocardial trabeculations orient toward the cardiac jelly and endocardial cells in the cardiac lumen (Manasek, 1968; von Gise and Pu, 2012) (Figure 1). Later on in cardiac development, the progenitor cells found in the linear heart tube will coalesce into the left ventricle of the mature four-chambered heart. The pharyngeal and splanchnic mesoderm forms the SHF. These cells will then migrate into the developing heart and give rise to the right ventricle, outflow tract, and portions of the inflow tract

(Buckingham et al., 2005) (Figure 2).

A B

Lumen

Endocardium

Cardiac Jelly

DAPI

Myocardium Myocardium E8.5 E10.0 Endocardium

Figure 1: Formation of trabeculations from the luminal surface of the primitive heart tube. A. The nascent heart is constituted of an inner endocardial cell layer separated from an outer myocardial layer by extracellular matrix proteins termed the cardiac jelly. B. During cardiogenesis, around E10.0, the myocardial trabeculations form from the luminal surface of the primitive heart. . 13

Figure 2: Diagram of Early Heart Development from E7.5-E10.5. FHF progenitors coalesce along the midline (green) forming the primitive linear heart tube and ultimately the majority of the cells of the left ventricle (LV) and inflow tract (IFT). Pharyngeal and splanchnic mesoderm form the SHF (red) and these cells will migrate into the developing heart and give rise to the right ventricle, outflow tract, and portions of the inflow tract. Image modified from Nature Reviews Genetics 6, 826-837 (November 2005). 14

A number of genetic defects in the endocardium (Grego-Bessa et al., 2007; Liu et al., 2010) and cardiac jelly (Camenisch et al., 2000) have resulted in abnormal tissue architecture of the early mouse heart leading to embryonic lethality. Mutations in hyaluronan synthase-2 (Has2) for example, cause a loss of hyaluronic acid (HA) in the cardiac jelly, embryonic lethality at midgestation, and a lack of myocardial trabeculation

(Camenisch et al., 2000). Likewise there is an increasing body of evidence which suggests that oriented cell division is essential for establishing proper tissue architecture of the developing heart (Meilhac et al., 2004). In addition, late in heart development, oriented mitotic divisions of epicardial cells also controls epicardial cell migration and contribution to the myocardium (Wu et al., 2010). However despite this evidence arguing for oriented cell division during early cardiogenesis, the underlining molecular mechanisms which regulate proper spindle positioning in early cardiac development and trabecular formation remain poorly understood.

Cell Polarity During Embryogenesis

Cell polarity is an essential and highly conserved component of all eukaryotic cells during tissue development and refers to the polarized organization of cell membrane associated proteins as well as the asymmetric organization of organelles and cytoskeleton. (Bryant and Mostov, 2008). Recent studies in mammalian tissue culture cells suggest that polarized cell divisions rely on the unequal distribution and segregation of key polarity proteins during mitosis. These proteins govern the generation and proper axis alignment of differentiated cell types during organogenesis. 15

During embryo development, polarity proteins regulate normal cellular physiology as well as tissue homeostasis and morphogenesis. Consequently absence of cell polarization has shown to have detrimental consequences to the developing tissue including tissue disorganization and tumorigenesis (Gonzalez, 2007; Knoblich, 2010;

Martin-Belmonte and Perez-Moreno, 2012).

The Par Polarity Complex

Cell polarity and spindle orientation are coupled through the Par polarity complex. The Par complex is composed of three proteins, Par3, Par6, and protein kinase

C iota (Prkci). First discovered during embryogenesis of C. elegans where polarity gene mutants caused loss of normal blastomere asymmetry and subsequent division cleavage planes (Watts et al., 1996), this complex controls the cell polarity necessary for normal tissue generation and morphogenesis.

After establishment of the Par complex to one cell pole, Par3 interacts with the adapter protein Inscuteable (Insc in mammals) which binds directly to Pins (partner of

Insc, homologue of vertebrate LGN/Gpsm2 and AGS3/Gpsm1). Pins then associates with the heterotrimeric G proteins (Gαi) and NuMA. Critically, NuMA interacts directly with the cell spindle to control the orientation and of the spindle and the division plane of mitotic cells leading to normal tissue development (Siller and Doe, 2009) (Figure 3).

Figure 3: Partitioning (Par) complex proteins. Polarized cell division is regulated by proteins asymmetrically distributed during mitosis: The protein complexes of Par3-Par6- Prkci and Gαi-LGN-NuMA, which govern the division plane of mitotic cells.

Upstream Polarity Signaling Cues

Cell polarity has been shown to be regulated by a variety of both intrinsic as well as extrinsic signaling cues. An example of polarizing intrinsic signaling cues would be asymmetric localization of cell-cell junctions which is used to denote cell polarity localization and subsequent polarized mitotic divisions (Prehoda, 2009). On the other hand, ligand signaling from nearby cells is a type of extrinsic polarization cue. An example of this type of signaling would be from interactions between a daughter cell and other neighboring cells that localize the polarity complex machinery to one pole of the cell and therefore control the specification of daughter cell fate (Prehoda, 2009).

Function of the Par Complex in Multicellular Organisms

Current research has shown this complex to be conserved in all multicellular organisms and is required for a number of polarized processes including asymmetric cell division (Betschinger et al., 2003), cell migration (Etienne-Manneville and Hall, 2003), and tight junction formation (Suzuki et al., 2001). Loss of function of any one of the Par complex genes has been shown to result in a number of cellular defects. These include severe changes in cellular homeostasis as well as abnormal spindle orientation during asymmetric divisions leading to loss of tissue organization and complete failure of correct organogenesis (Silk et al., 2009).

For example in the neuroblast of C. elegans and D. melanogaster, Par cortical proteins align the mitotic spindle along defined axes (Siller and Doe, 2009). Likewise the zebra fish Heart and Soul gene encoding the atypical protein kinase C, directs the apical 18 targeting of the zonula adherens. Mutations in this gene result in defects in heart tube assembly as well as the disruption of polarized epithelia in the retina, neural tube, and digestive tract (Horne-Badovinac et al., 2001).

Symmetric Polarized Divisions vs. Asymmetric Polarized Divisions

During organogenesis of the Drosophila neuroblasts, cells use Par polarity for surprisingly diverse functions. Here, polarized stem cells divide either symmetrically or asymmetrically to generate progeny with two different fates (Figure 4). The major purpose of symmetric polarized divisions is cell proliferation and expansion. During symmetric polarized cell division, one polarized daughter cell produces two identical daughter cells which will both acquire the same developmental fate and are indistinguishable from the mother cell. This therefore maintains and populates the current nervous system pool through self-renewal.

On the other hand, the major purpose of asymmetric polarized divisions is cell differentiation. During an asymmetric polarized cell division, the other daughter cell will give rise to two cells with different developmental fates depending on the difference in polarity protein expression levels between the two daughter cells. In this case, one daughter will differentiate along a specific lineage and therefore have less potency than the mother cell. The other cell however will maintain its potential to self-renewal similar to the mother cell (Prehoda, 2009). This occurs in the Drosophila testis where the germ stem cell divides perpendicular to a niche structure termed the hub. This 19 ensures that one cell will continue as a stem cell attached to the hub, while the other differentiates into a gonial blast (Yamashita et al., 2003).

Figure 4: Symmetric vs. Asymmetric cell Division in Epithelial Cells. A. During a symmetric polarized division, spindle orientation is parallel to the Par polarity machinery (red). Thus the complex will segregate equally and give rise to two equal cells executing a cell self-renewal program. B. During an asymmetric polarized division, spindle orientation is perpendicular to the Par polarity machinery. Thus the complex will segregate unequally and give rise to a differentiating cell. Therefore, the difference in polarity protein expression levels between two daughter cells mediates an asymmetric cell division.

The Protein Kinase C Family

The protein kinase C (PKC) family of serine-threonine kinases consists of 9 different genes giving rise to at least 12 isoforms. These isoforms can be further subdivided into 3 subfamilies. The different subdivisions are based on their dependency on cofactors during their activation process. First the classical PKCs (α, β and γ) are dependent on Ca2+ions as well as diacylglycerol for their activation. On the other hand, the novel PKCs (δ, ε, η and θ) are Ca2+ ion independent but still require diacylglycerol for their activation. Finally the subfamily of atypical PKCs (ζ and ι/λ,) are independent of

Ca2+ ions as well as diacylglycerol for their activation (Seidl et al., 2013).

Essential Role of Prkci During Embryogenesis

The atypical PKCs represent a unique as well as important subgroup among the entire PKC family. Complete Prkci null mouse embryos die by day 9.5 and are morphologically abnormal lacking in any recognizable structures (Soloff et al., 2004). In the developing eye, a Prkci conditional knock out mouse line where the Prkci gene is inactivated in postmitotic photoreceptors caused severe laminar disorganization throughout the entire retina (Koike et al., 2005). Even in the immune system, disruption of Prkci in activated T cells caused severe defects in Th2 cytokine production. When challenged with an allergic stimulus, Prkci mutant mice had an impaired lung inflammatory response (Yang et al., 2009).

Additionally the other members of the Par polarity complex have also been shown to be essential during mammalian embryogenesis as well. Previous studies have 22 demonstrated essential roles of the Par complex in regulating stratification and differentiation of mammalian skin (Lechler and Fuchs, 2005), development of gut epithelium (Goulas et al., 2012), and eye lens formation (Sugiyama et al., 2009).

However despite an extensive body of literature documenting the importance of the distribution of cell polarity proteins and polarized cell division during organogenesis, the role of these cellular processes in normal cardiac development and trabecular formation has not been adequately examined in the developing heart. Herein we sought to address this gap in knowledge and define the role of the Par complex in directing cell polarization in early cardiac morphogenesis and trabecular formation.

Hypotheses: Based on the previous discussion, the following hypotheses were developed.

1. Par complex proteins are asymmetrically localized in the developing

myocardium.

2. Par polarity proteins co-localize together in the developing myocardium.

3. Cardiac jelly mediates the localization of polarity complex to one pole of

the cardiomyocyte.

4. Par complex proteins regulate oriented cellular divisions of mitotic

cardiomyocytes in the early developing heart.

5. Disruption of the Par complex leads to disruption of normal

cardiogenesis.

6. Ex vivo embryonic cardiomyocytes can spatially polarize in vivo.

Taken together, these data would provide evidence for the following hypothetical scheme (Figure 5).

Figure 5: Hypothetical Representation of the Spatial Organization of the Early Embryonic Heart. A. During the linear heart tube stage of heart development, the nascent heart consists of layer of myocardium separated from the endocardium by a layer of extracellular matrix called cardiac jelly. B. We hypothesize that signals originating in the cardiac jelly polarize the layer of myocardium C, and this leads to the normal heart development including trabeculations that orient inward towards the heart’s lumen D. 25

CHAPTER II: MATERIALS AND METHODS 26

Immunohistochemistry

Embryos were fixed in 4% paraformaldehyde for 12-15 minutes and flash frozen in OCT, then stored at -80°C until cryosectioning. The sections were rinsed once in PBS for 10 minutes and blocked in 10% donkey serum in PBS/0.1% saponin. Primary antibodies were incubated with the sections overnight at 4°C. Following three 20 minute

PBS washes, secondary antibodies with conjugated fluorochromes (Invitrogen) were added to each section for 60 min at 37°C. The sections were then washed three times for 20 minutes in PBS and then mounted using Prolong® antifade mounting medium

(Invitrogen).

Primary Antibodies used were as follows: anti-Pard6g, anti-Prkci, anti-Pard3, and anti-gamma tubulin (Santa Cruz Biotechnology). anti-giantin, anti-phospho histone H3, anti-pericentrin, anti-Ki67 (abcam). anti-Troponin T (Pierce). anti-CD31 (BD

Pharmingen). anti-α actinin (Sigma-Aldrich). anti-survivin (Cell Signaling), anti-NuMA was a kind gift from Duane A. Compton, Department of Biochemistry, Dartmouth Medical

School.

Animal breeding

All mouse related studies were approved by the Subcommittee on Research

Animal Care at Massachusetts General Hospital. To obtain mouse embryos that are cardiac deficient in Prkci or NuMA expression, we interbreed the previously described

Nkx2.5+/Cre with either Prkci Flox/Flox (a kind gift from Shigeo Ohno at Yokohama City

University ) or NuMA Flox/Flox (a kind gift from Don W. Cleveland at UC San Diego School 27 of Medicine ), to generate mice harbouring either Nkx2.5 +/Cre/Prkci +/Flox or Nkx2.5

+/Cre/NuMA +/Flox alleles. We bread these mice with mice carrying Prkci Flox/Flox alleles or

NuMA Flox/Flox respectively.

We also bred HAS2 null (Camenisch et al., 2000) and CD44 null(Protin et al.,

1999) mice to create HAS2 -/- and CD44-/- embryos respectively. This generated embryos that carry homozygous Prkci Flox/Flox alleles or NuMA Flox/Flox alleles and Nkx2.5 +/Cre. At days 8.5 and 10.5 post coitum, we euthanized the pregnant mother and dissected the embryos from the surrounding yolk sac and uterus. Embryos were subsequently imaged on a whole mount dissection microscope and their genotypes were determined by PCR analysis of tail biopsy samples.

Image analysis

Confocal 3D co-localization images were analyzed using PerkinElmer Volocity software. Image Z-stacks were first deconvolved using iterative restoration and then

Manders and Pearson correlation coefficients computed with Volocity software. Scatter plots were generated from unenhanced, deconvolved images.

Electron microscopy

E10.5 Embryos were prepared and cryosectioned for EM scanning as previously described (Camenisch et al., 2000). Images of adjacent immunostained myocardium were then overlaid onto the EM images.

Co-immunoprecipitation

Total protein from E9.0 embryos was isolated using IP lysis buffer (Pierce,

Rockford, IL). Co-immunoprecipitation was performed using the Pierce Co-

Immunoprecipitation Kit following manufacturer's protocol. Protein-antibody mixture was incubated o/n at 4 °C with 5 μg antibody against Pard6g or rabbit isotype (Santa

Cruz Biotechnology, Santa Cruz, CA). During the elution of the precipitated fraction, the samples were boiled for 5 min. at 100 °C in elution buffer with 6x loading buffer. Pull down samples and samples containing total lysate were loaded, separated, and transferred to a PVDF membrane using the Mini-Protean® Tetra system and the Mini

Trans-Blot® Cell (BioRad, Hercules, CA). Membranes were blocked in 5% non-fat dry milk in 0,1% TBST and incubated with primary antibodies against Prkci (BD Biosciences, San

Jose, CA). Subsequently, the membranes were probed with horseradish peroxidase- conjugated anti mouse (Jackson Immuno, West Grove, PA). The signal was visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). ImageJ software was used for analysis. qPCR

Total RNA of E10.5 wildtype and Prkci knockout hearts was isolated using the

RNeasy Mini Kit (Qiagen, Valencia, CA) according manufacturer's protocol. cDNA was made using iScriptTM cDNA Synthesis Kit (BioRad, Hercules, CA). Quantitive polymerase chain reaction was performed with SYBR GreenERTM qPCR SuperMix for ABI PRISM ®

(Invitrogen, Carlsbad, CA) and specific primers against β-actin, β-MHC, Nkx2.5, and the trabeculation markers BMP10, connexin40, and PEG1. 29

β-actin F 5’-GGCTGTATTCCCCTCCATCG-3’

R 5’-CCAGTTGGTAACAATGCCATGT-3’

β-MHC F 5’-ACTGTCAACACTAAGAGGGTCA-3’

R 5’-TTGGATGATTTGATCTTCCAGGG-3’

BMP10 F 5’-ATGGGGTCTCTGGTTCTGC-3’

R 5’-CAATACCATCTTGCTCCGTGAA-3’

Connexin40 F 5’-GGTCCACAAGCACTCCACAG-3’

R 5’-CTGAATGGTATCGCACCGGAA-3’

Nkx2.5 F 5’-GACAAAGCCGAGACGGATGG-3’

R 5’-CTGTCGCTTGCACTTGTAGC-3’

PEG1 F 5’-TGACCCTGAGGTTCCATCGAG-3’ R 5’-GCCGCAGAAGGGACTCTAC-3’ 30

Ex vivo scratch assay

For ex vivo cardiac cell cultures, E12.5 hearts from wild-type mice were digested for 1 hour in collagenase A and B (1 mg/ml) and then 3 minutes in .25% Trypsin-EDTA to a obtain single-cell suspension. The cells were seeded onto a pre-gelatinized 96 well plate in mouse differentiation media. After 24hours, the cells were scratched in the presence of DMSO or GF 109203X (R&D systems) according to the previous described protocols

(Etienne-Manneville and Hall, 2001; Liang et al., 2007) before fixation and immunostaining.

Measurements, quantification, and statistical analysis

qPCR results and the results related to the percentage of positive Ki67 and pHH3 cardiomyocytes in embryonic hearts are presented as mean ± SEM. Differences between groups were compared with a student's t test and one-way ANOVA with Dunnett post- hoc analysis respectively. A p value of <0.05 was considered statistically significant with

* P≤0.05, ** P≤0.01, and *** P≤0.001. The method for measurement of division angles has been described previously in (Lechler and Fuchs, 2005).

Briefly, early stage mitotic cells were identified by pHH3 immunostaining and the centromere was identified by Pcnt immunostaining. Late-stage mitotic cells were identified by the presence of survivin at the midbody/cleavage furrow. Cells were scored only if both daughter nuclei surrounding the survivin staining could be unambiguously identified. Angles were measured by drawing a line through the centromere (early mitosis) or the centers of the two nuclei (late stage mitosis), and compared relative to 31 the cardiomyocyte luminal membrane. Images were captured and measurements were quantified blind to genotype.

To reduce any bias in data collection, all data from each group were not analyzed until all images were collected. n values indicated in each radial histogram indicate the number of luminal myocardial cells collected from 3-5 embryos. No statistical method was used to predetermine sample size, but data was collected from all available embryos of the indicated genotypes.

Radial histograms were generated using Origin 9.0 software from data binned into 10° increments from 0 to 90°. For assessment of statistical significance of spindle orientation and angle of cell division, a Chi-square test was calculated using two degrees of freedom. Data was categorized as follows: perpendicular (70-90°); planar (0--

20°); oblique (20°-70°). As opposed to having equal 30° bins, we used these three categories based on the observation that most cardiomyocyte cell divisions fell into these ranges in wild type E8.5 hearts. Therefore a 20°-70° range represents a better deviation from normal than a 30°-60° range would.

Data sets were compared by chi-square tests shown in Table 1 as previously described in (Williams et al., 2014). No statistical differences were found between age- matched littermates. All other graphs and statistical analyses (Student’s t-tests and chi- square tests) were produced using GraphPad Prism.(Prehoda, 2009)

CHAPTER III: RESULTS 33

Par Complex components localize asymmetrically in luminal myocardial cells.

We first examined whether polarity proteins are asymmetrically localized in the linear heart tube, before the onset of trabeculation. Immunohistochemistry of E8.5 mouse embryos showed that both Prkci and its interacting partner Pard6g are localized to the luminal side of luminal Troponin T (TnT) positive myocardial cells of the outflow tract (Figure 6A), common ventricle (Figure 6B), and sinus venosus (Figure 6D).

We then demonstrated that Prkci and Pard6g co-localized together with confocal microscopy (Figure 7A and 7B). Finally we confirmed that Pard6g and Prkci interact in a complex by co-immunoprecipitating Pard6g from embryonic lysates and immunoblotting for Prkci (Figure 7C).

Figure 6: Luminal localization of Par complex members throughout the early embryonic heart. The outflow tract A, common ventricle B, and sinus venosus C, of an E8.5 embryo co-stained for Pard6g (green), Prkci (red), Troponin (purple) and DAPI (blue). Scale bars: 100mzoom out, 10melsewhere. E8.5 mouse diagram above each panel represents tissue section level within the mouse heart (red line).

Figure 7: Luminal co-localization of Pard6g and Prkci in early embryonic hearts. A. Confocal microscopy showing co-localization of Pard6g (green) and Prkci (red). B. A 2D scatter plot of fluorescence intensities in the green (x, Prkci) and red (y, Pard6g) channels was reconstructed from the 3-D image and the Manders Overlap Coefficient (MOC) and Pearson Correlation Coefficient (r) were calculated. This demonstrates significant co-localization of the red and green channels in three dimensions. C. Embryonic lysates were immunoprecipitated with anti-Pard6g antibody and then immunoblotted with anti-Prkci. TnT (purple) and DAPI (blue). Scale bars: 10μm. 36

Next we systematically investigated whether other members of the polarity complex are also restricted to the luminal side of myocardial cells. As shown in Figure

8A-M and Figure 9A-F, members of the polarity complex are localized in a spatially restricted pattern to the luminal side of luminal myocardial cells and are co-localized with each of the other complex members. These results are consistent with findings from multiple laboratories (Betschinger et al., 2003; Lechler and Fuchs, 2005; Shi et al.,

2003) examining different organ systems, demonstrating that the Par polarity proteins interact as part of a cell polarization complex.

Figure 8: Luminal localization of Par complex members in early embryonic hearts. A-M. Par complex members co-stained green or red as indicated. TnT (purple) and DAPI (blue). Scale bars: 10μm.

Figure 9: Confocal microscopy images of co-localization of Par complex members. A-F. Confocal microscopy depicting the co-localization of specified polarity proteins on E8.5 ventricular tissue. TnT (purple), DAPI (blue). Scale Bar: 10m. 39

Luminal myocardial cells undergo polarized cell division in the early developing heart.

Previously Le Garrec et al. demonstrated that myocardial cell division is oriented with a planar bias in embryonic mouse hearts (Le Garrec et al., 2013). We therefore examined E8.5 embryos (prior to the onset of trabeculation) to determine whether luminal myocardial cells displayed oriented cell division relative to the axis of polarity defined by Par complex localization.

Prior work by Lechler and Fuchs established a standard methodology for the analysis of cell division angles relative to the basement membrane (Lechler and Fuchs,

2005). This approach also accounts for the three dimensional nature of divisions within tissue and excludes division outside the plane of the section or those occurring into the

Z- plane. We adapted this approach to analyze the mitotic division plane of both early

(Figure 10) and late (Figure 11) mitotic luminal ventricular cardiomyocytes relative to the cardiac lumen.

To examine the plane of cell division of cardiomyocytes in the early stages of mitosis, E8.5 hearts were co-stained for TnT (cardiomyocyte marker), pericentrin (Pcnt, centromere marker), phospho-Histone H3 (pHH3, early mitosis marker), and DAPI (DNA stain). We were thereby able to define the spindle alignment and cell division plane of dividing cardiomyocytes relative to the cardiac lumen. As shown in Figures 10A and 10B, most early mitotic cell divisions occurred in a division plane parallel to the heart lumen.

Of great interest however, a minority of mitotic cells had an alignment that was perpendicular to the lumen. 40

Figure 10: Polarized cell division in early mitotic luminal myocardial cells of E8.5 hearts. A. Top panels: Representative images of early mitotic luminal cardiomyocytes in E8.5 wild-type hearts co-stained for pHH3 (green), Pcnt (yellow), TnT (purple), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in early-stage mitotic cells from E8.5 wild-type hearts. Scale bars: 10μm. 41

pHH3 identifies predominantly late prophase, pro-metaphase, and metaphase cells.

Since the early mitotic spindle can rotate during this window of time, we repeated the assay focusing solely on telophase cells in which both spindle poles can be readily identified within a thin section. Coalesced survivin immunostaining was used to identify the cleavage furrow of telophase cells and the reference line was drawn using the center of the segregating DNA mass when the spindle is in its committed orientation. As shown in Figures 11A and 11B, we again observed most late mitotic cell divisions occurring in a parallel division plane relative to the heart lumen, while a small population of dividing cells had an alignment that was perpendicular to the heart lumen.

These results are reminiscent of cell division in murine skin cells where a shift from predominantly parallel/symmetric to perpendicular/asymmetric cell division coincides with epidermal stratification (Lechler and Fuchs, 2005).

Figure 11: Polarized cell division in late mitotic luminal myocardial cells of E8.5 hearts. A. Top panels: Representative images of luminal telophase cardiomyocytes in E8.5 wild-type hearts co-stained for survivin (green), TnT (red), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). D. Radial histogram depicting the orientation of cardiomyocyte divisions in late-stage mitotic cells from E8.5 wild-type hearts. Scale bars: 10μm.

Hyaluronic Acid (HA) in the cardiac jelly is required for Par complex localization.

Our findings suggest that myocardial cells within the nascent heart are able to orient with respect to the lumen and raises the possibility that cardiac jelly mediates trabecular formation by directing the polarization and asymmetric cell division of luminal myocardial cells. Mutations in hyaluronan synthase-2 (Has2) result in loss of HA in the cardiac jelly, embryonic lethality at midgestation, and a lack of myocardial trabeculation (Camenisch et al., 2000). We therefore bred the Has2 null mouse line to create Has2–/– embryos (Camenisch et al., 2000). E8.5 Has2–/– embryos had total delocalization of the Par complex from the luminal myocardium (Figure 12A-D). In addition, the luminal myocardial cells of the mutant embryo had a random pattern of cell division orientation during both early (Figure 13) as well as late (Figure 14) mitosis.

Figure 12: HA in the cardiac jelly is required for Par complex localization in E8.5 mouse hearts. A. E8.5 HAS2-/- embryo co-stained for TnT (purple), PECAM-1 (yellow), and DAPI (blue). B-D. E8.5 HAS2-/- embryonic hearts stained in green for Prkci (B), Pard6g (C), or NuMA (D). E. E8.5 HAS2+/+ littermate control embryo co-stained for TnT (purple), PECAM-1 (yellow), and DAPI (blue). F-H. E8.5 HAS2+/+ littermate control embryonic hearts stained in green for Prkci (B), Pard6g (C), or NuMA (D). Scale bars: 100mzoom out, 10melsewhere.

Figure 13: HA in the cardiac jelly is required for oriented cell division in early mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of early mitotic luminal cardiomyocytes in E8.5 HAS2-/- hearts co-stained for pHH3 (green), Pcnt (yellow), TnT (purple), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in early-stage mitotic cells from E8.5 HAS2-/- hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between HAS2-/- and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Figure 14: HA in the cardiac jelly is required for oriented cell division in early mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of luminal telophase cardiomyocytes in E8.5 HAS2-/- hearts co-stained for survivin (green), TnT (red), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in late-stage mitotic cells from E8.5 HAS2-/- hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between HAS2-/- and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Although HA has been shown to bind to the CD44 receptor (Banerji et al., 1999),

CD44–/– mice are viable with no trabeculation or other overt cardiovascular phenotype

(Schmits et al., 1997). Nonetheless, we examined whether CD44–/– mice have defects in

Par complex localization and spindle orientation in E8.5 embryos. As expected, Par complex localization and spindle orientation were not altered (Figures 15-17). We therefore conclude that HA acts indirectly to control polarized cardiomyocyte division, potentially as a co-regulator facilitating endocardial signalling cues.

Figure 15: Figure 3: CD44 is not required for polarized cell division in E8.5 mouse hearts. A. E8.5 CD44-/- embryo co-stained for TnT (purple), PECAM-1 (yellow), and DAPI (blue). B-D. E8.5 CD44-/- embryonic hearts stained in green for Prkci (J), Pard6g (K), or NuMA (L). Scale bars: 100mzoom out, 10melsewhere.

Figure 16: CD44 is not required for oriented cell division in early mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of early mitotic luminal cardiomyocytes in E8.5 CD44-/- hearts co-stained for pHH3 (green), Pcnt (yellow), TnT (purple), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in early-stage mitotic cells from E8.5 CD44-/- hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between CD44-/- and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Figure 17: CD44 is not required for oriented cell division in late mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of luminal telophase cardiomyocytes in E8.5 CD44-/- hearts co-stained for survivin (green), TnT (red), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in late-stage mitotic cells from E8.5 CD44-/- hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between CD44-/- and wildtype cells. Chi- square P values related to B are in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals. 51

Prkci is required for polarized cell division, ventricular trabeculations, and embryonic viability.

Next we adopted a genetic approach to directly test whether cell polarity is required for trabecular formation. We bred a mouse line harboring a Prkci gene in which the essential exon 5 is flanked by loxP sites (Sugiyama et al., 2009) with a knock-in mouse line expressing Cre under the control of the mouse Nkx2.5 promoter (Moses et al.,

2001). Myocardial deletion of the Prkci gene was embryonically lethal with no viable pups born (Figure 18). Gross analysis of the E10.5 knockout mouse heart revealed no grossly apparent morphological changes (Figure 19). Staining of E10.5 embryos for TnT,

PECAM-1, and DAPI demonstrated a trabeculation defect with no trabeculations observed in the cardiac null mouse heart. In addition, the endocardium in the cardiac conditional null had no apparent morphological defects compared to the control embryo (Figures 20 & 21).

Figure 18: Genotypes of pups born from an Nkx 2.5+/cre/Prkci+/fl male crossed with a Prkcifl/fl female. Graph showing observed and expected genotypes of pups born from an Nkx2.5+/cre/Prkci+/fl male crossed with a Prkcifl/fl female.

Figure 19: Gross morphological analysis of an E10.5 Prkci cardiac null mouse heart. External structures of the heart from an E10.5 wild-type (A) and an E10.5 Prkci cardiac null embryo (B). Scale bars: 1mm.

Figure 20: Prkci is required for normal ventricular trabeculation. A&B. E10.5 littermate control (A) and Prkci cardiac conditional knockout (cKO) (B) embryos immunostained for PECAM-1 (yellow), TnT (red), DAPI (blue). Scale bars: 100m.

Figure 21: Comparison of endocardium from WT and Prkci cardiac null E8.5 embryos. IHC images stained for DAPI (blue), PECAM-1 (green), and TroponinT (red) showing no difference in the endocardial morphology between the specified E8.5 embryos. Scale Bar: 100m.

To further delineate the trabeculation defect, we imaged the heart with a combination of scanning electron microscopy (EM) and immunohistochemistry. Prkci cardiac null and control embryos were cryosectioned to the level of the mid cardiac cavity. The ultrastructure of the embryonic heart was then examined with scanning EM. The adjacent cryosections were immunostained for TnT and the immunostained images were then overlaid with the scanning EM images (Figure 22). As shown, this allowed us to demonstrate the absence of ventricular trabeculation in E10.5 conditional null hearts compared to the control. We then confirmed that the trabecular markers BMP10,

Cnx40, and PEG1 were down regulated in the Prkci mutant heart, however the pan- cardiac markers Nkx2.5 and -MHC were unchanged (Figure 23).

Figure 22: Scanning electron microscopy and immunohistochemistry of the E10.5 Prkci conditional null heart. A-D. Scanning EM of cryosectioned E10.5 littermate heart (A) or Prkci null heart (B). EM images are overlaid with adjacent TnT immunostained sections (C&D).

Figure23: RT-PCR analysis in E10.5 WT and Prkci mutant hearts. P values (*P≤0.05, ** P≤0.01, and *** P≤0.001.) determined by a Student’s t-tests. 59

Prior work from the Pu laboratory demonstrated that the Nkx2.5-Cre mouse line can result in LoxP excision in the myocardium alone (in e.g. the Rosa26 locus) or in both the myocardium and endocardium (in e.g. the GATA4 locus) (Ma et al., 2008). To determine if this mouse line resulted in Prkci deletion in both the endocardium and the myocardium or in the myocardium alone, we immunostained the Prkci cardiac null embryos for Prkci. E8.5 Nkx-Cre/Prkci embryos and somite-matched controls were cryosectioned and stained with Prkci, PECAM-1, TnT, and DAPI. As shown in Figure

24A&B, Prkci staining was absent from the mouse myocardium but present in the endocardium in cardiac null embryos, therefore validating the specificity of the Prkci antibody and demonstrating the myocardial specific deletion of Prkci.

In addition we also stained for Par complex members Pard6g and NuMA that we found to be delocalized only within the myocardium of the Prkci cardiac null hearts

(Figure 24C-F, Figure 25).

Figure 24: Localization of Par complex members in Prkci cardiac null. A-F. Immunostaining of littermate control (A, C, E) or Prkci cardiac null (B, D, F) E8.5 embryos stained for Prkci (A&B), Pard6g (C&D), or NuMA (E&F). Specified polarity protein (green), DAPI (blue), PECAM-1 (red), and TnT (purple). Closed arrowheads point to faint polarity protein staining on the endocardium. Open arrowheads point to luminal polarity protein staining on myocardium. Scale bars: 10μm.

Figure 25: Rosa mT/mG crossed with Nkx2.5+/cre and Prkci fl/fl . A. External structure of WT E10.5 embryo in an RosamT/mG background. B. Cryosection of WT E10.5 embryo from previous image. C&D. Immunostaining of littermate control E10.5 embryos in a RosamT/mG background stained for Prkci (C) or Pard6g (D). E. External structure of KO E10.5 embryo in a RosamT/mG background. F. Cryosection of KO E10.5 embryo from previous image. G&H. Immunostaining of a Prkci cardiac null E10.5 embryos in a RosamT/mG background stained for Prkci (G) or Pard6g (H). Specified polarity protein (purple), DAPI (blue), and Nkx.2.5+/cre cells are eGFP+ (green).

Since Prkci is an integral component of the cell polarity machinery, we then examined mitotic division planes in Prkci cardiac null embryos during early and late stages of mitosis. As shown, deletion of Prkci resulted in a loss of perpendicular cell divisions relative to the heart’s lumen during both early (Figure 26) and late (Figure 27) stages of mitosis .

Previous work with a variety of cell types has shown that a scratch injury of a confluent monolayer of cells initiates directional cell migration and Golgi reorientation perpendicular to the wound (Etienne-Manneville and Hall, 2001). To determine if myocardial cells are capable of polarization during in vitro culture, we performed a scratch-wound assay on cardiomyocytes isolated from E12.5 mouse hearts. As shown in

Figure 28A&B, in response to the scratch, embryonic cardiomyocytes at the leading edge reoriented their Golgi apparatus to face the wound.

We then repeated the experiment with GF109203X, a small molecule inhibitor of atypical protein kinase C (Uberall et al., 1999). As shown in Figure 28C&D, this prevented Golgi reorientation towards the wound edge.

These results show that embryonic cardiomyocytes cultured in vitro can be induced to polarize and that this polarization is dependent on atypical protein kinase C activity. Collectively our results demonstrate that Prkci is required for polarized Par complex localization, correct spindle orientation, and normal ventricular trabeculation.

Figure 26: Prkci is required for oriented cell division in early mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of early mitotic luminal cardiomyocytes in E8.5 Prkci cardiac null hearts co-stained for pHH3 (green), Pcnt (yellow), TnT (purple), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in early-stage mitotic cells from E8.5 Prkci cardiac null hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between Prkci null and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Figure 27: Prkci is required for oriented cell division in late mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of luminal telophase cardiomyocytes in E8.5 Prkci cardiac null hearts co-stained for survivin (green), TnT (red), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in late-stage mitotic cells from E8.5 Prkci cardiac null hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi- square test in B for determining significance of the difference between Prkci null and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Figure 28: Atypical protein kinase C activity is required to induce polarization of embryonic cardiomyocytes cultured in vitro. A. Representative example of Golgi reorientation 16hrs after scratch injury of a confluent layer of E12.5 embryonic cardiomyocytes cultured in vitro. B. Percentage of leading edge cardiomyocytes with the Golgi in the 90° sector facing the scratch injury. C. Representative example of Golgi reorientation 16hrs after wounding in vitro E12.5 cardiomyocytes in the presence of GF109203X. D. Percentage of leading edge cardiomyocytes with the Golgi in the forward-facing 90° sector in the presence GF109203X. Alpha-actinin (green), Golgi (red), DAPI (blue). Scale bars: 10μm.

NuMA is required for polarized cell division, ventricular trabeculations, and embryonic viability.

Our results show that deletion of HA in the cardiac jelly or Prkci in the developing myocardium results in a loss of cell polarity, loss of normal perpendicular cell division, and defective trabeculation. NuMA acts downstream of Prkci and directly binds dynein to orient the cell spindle and mitotic division plane (Hawkins and Garriga, 1998; Silk et al., 2009). We therefore bred a conditional null NuMA mouse line (Silk et al., 2009) with the Nkx2.5-Cre mouse line to generate cardiac null NuMA mouse embryos.

Myocardial specific deletion of the NuMA gene was embryonically lethal with no viable pups born (Figure 29). Gross analysis of the E10.5 NuMA cardiac null mouse heart revealed no revealed no abnormal changes to the heart’s morphological structure

(Figure 30).

Immunohistochemical staining of E10.5 embryos for TnT and PECAM-1 demonstrated that the NuMA cardiac null, like the Prkci cardiac null, had a trabeculation defect while the endocardium appeared morphologically intact (Figures 31 & 32).

Figure 29: Genotypes of pups born from an Nkx 2.5+/cre/NuMA+/fl male crossed with a NuMAfl/fl female. Graph showing observed and expected genotypes of pups born from an Nkx2.5+/cre/NuMA+/fl male crossed with a NuMAfl/fl female. 68

Figure 30: Gross morphological analysis of an E10.5 NuMA cardiac null mouse heart. External structures of the heart from an E10.5 NuMA cardiac null embryo. Scale bars: 1mm.

Figure 31: NuMA is required for normal ventricular trabeculation. A&B. E10.5 somite matched (SM) control (A) and NuMA cKO (B) embryos immunostained for PECAM-1 (yellow), TnT (purple), DAPI (blue). Scale Bar: 100mm. 70

Figure 32: Comparison of endocardium from WT and NuMA cardiac null E8.5 embryos. IHC images stained for DAPI (blue), PECAM-1 (green), and TroponinT (red) showing no difference in the endocardial morphology between the specified E8.5 embryos. Scale Bar: 100mm.

As expected, immunostaining of NuMA was absent from the myocardium in the

NuMA cardiac conditional null (Figure 33A and B). In contrast, Prkci localization was not altered in the cardiac null, consistent with NuMA being downstream of Prkci (Figure 33C and D).

Of note, we did not observe any significant differences in cardiomyocyte proliferation in cardiac null NuMA embryos or the other mutant embryos compared to controls (Figure 34A and B).

We then examined whether early and late cardiomyocyte divisions are altered in the NuMA cardiac null. As shown, there was a marked reduction of perpendicular cell divisions and an increase in oblique and parallel cell divisions relative to the heart’s lumen in both the early (Figure 35) and late (Figure 36) mitotic cardiomyocytes.

Accordingly, we conclude that NuMA directs trabecular formation by orienting the cell spindle and cell division plane perpendicular to the nascent heart’s lumen.

Figure 33: NuMA is required for cell polarization and normal ventricular trabeculation. A-D. Immunostaining of a littermate control (A and C) or NuMA cardiac null (B and D) embryo stained for NuMA (A and B) or Prkci (C and D). Specified polarity protein (green), DAPI (blue), and TnT (purple). Scale bars: 10μm.

Figure 34: HAS2, Prkci, or NuMA nulls do not alter cell prolfieration rates in E8.5 embryos. Quantification of Ki67 (A) or pHH3 (B) positive cardiomyocytes in specified E8.5 mutant embryos. NS, not significant.

Figure 35: NuMA is required for oriented cell division in early mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of early mitotic luminal cardiomyocytes in E8.5 NuMA cardiac null hearts co-stained for pHH3 (green), Pcnt (yellow), TnT (purple), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in early-stage mitotic cells from E8.5 NuMA cardiac null hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between NuMA null and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Figure 36: NuMA is required for oriented cell division in late mitotic E8.5 cardiomyocytes. A. Top panels: Representative images of luminal telophase cardiomyocytes in E8.5 NuMA cardiac null hearts co-stained for survivin (green), TnT (red), and DAPI (blue). Lower panels: Superimposed axis of cell division (solid line) relative to the cardiac lumen (dashed line). B. Radial histogram depicting the orientation of cardiomyocyte divisions in late-stage mitotic cells from E8.5 NuMA cKO hearts. Scale bars: 10m. P values (P≤0.001 for early and late mitosis) determined by a chi-square test in B for determining significance of the difference between NuMA null and wildtype cells. Chi-square P values related to B are shown in Table 1 and Table 2. n values in B represent the number of cells analyzed from 3 to 5 independent animals.

Lineage tracing of ventricular trabeculations

These results are consistent with the hypothesis that individual trabeculations are derived from cardiomyocytes undergoing perpendicular cell division and suggest that individual trabeculations may be derivative from the same mother cell. To test this, we turned to the multicolor reporter system of the R26R-Confetti 2.1 mouse line wherein

Cre-mediated recombination results in one of four ﬂuorescent markers being randomly placed under control of the constitutive CAGG promoter (Snippert et al., 2010). We bred the R26R-Confetti mouse line to the Nkx2.5-cre mouse line. Two-photon excitation microscopy of E10.5 transgenic hearts revealed that cells of an individual ventricular trabeculation were of the same color, consistent with them being derived from a single mother cell (Figures 37 and 38).

Figure 37: Polarized cell division and perpendicular spindle orientation direct trabecular formation. A. Two-photon excitation microscopy of E10.0 embryonic R26R+/Confetti /NKX2.5+/Cre hearts. Representative examples of a single yellow (A), single blue (B), single red (C) trabeculation arising from multicolored compact myocardium. D. Percentage of cells that are one (uni), two (double), or three (tri) colors in a 100umx100um area in the compact versus the trabecular myocardium. Scale bars: 10m.

Figure 38: Confocal microscopy of an E9.5 Nkx 2.5+/cre R26R+/Confetti transgenic heart. A, Image showing single-color trabeculations arising from multicolor compact myocardium. B-D, 2D confocal microscopy images depicting single-color trabeculations arising from multicolour compact myocardium. Scale bars: 200m and 10m.

Table 1: Chi-square test values for spindle orientation and cell division plane in WT vs. KO embryos. Cells were categorized as perpendicular (70-90°), planar (0-20°), or oblique (20°-70°). No statistical differences were ever found between age-matched littermates. NS, not significant.

Table 2: Chi-square test values for spindle orientation and cell division plane in early mitotic vs. late mitotic myocardial cells. Cells were categorized as perpendicular (70-90°), planar (0-20°), or oblique (20°-70°). No statistical differences were ever found between age-matched littermates. NS, not significant.

CHAPTER IV: DISCUSSION 82

Summary and Main Experimental Findings

Myocardial trabeculation is a key morphologic event during cardiogenesis.

Although a number of signaling molecules have been implicated in trabeculation (Grego-

Bessa et al., 2007; Liu et al., 2010), the cellular processes that are required for trabecular formation are not fully understood in mammals. The aim of this present study was to shed light on early embryonic in vivo functions of cell polarity in the mouse heart. Herein we show that in mammals, polarized cell division is required for myocardial trabeculation and normal cardiogenesis.

Polarity during embryogenesis

Throughout evolution, from bacteria to worms to mammals, cell polarity plays a central role in development and function. How individual cells are organized and how this organization leads to the assembly of complex organs is a fundamentally important and deeply intuitive question. Earlier genetic studies in C. elegans, Xenopus and

Drosophila melanogaster demonstrated an essential and pivotal role of Prkci in the establishment and maintenance of polarity of oocytes and epithelial cells (Cha et al.,

2011; Dominguez et al., 1992; Izumi et al., 1998; Tabuse et al., 1998). We therefore hypothesized that cell polarity is an essential component to the normal development of the mammalian heart during early cardiogenesis.

The Heart Polarity Model

The Par3/Par6/Prkci complex is required for the regulation of polarity in a variety of cells in all multicellular organisms. The protein Prkci forms a complex with Par6 as well as Par3 and contributes to the cell polarity in various biological contexts. Here we show that key members of the Par complex, including Prkci, are localized to the luminal side of the luminal myocardial cells and this localization requires a normal composition of the cardiac jelly.

Cardiac Jelly and Myocardial Polarity

Hyaluronic acid is a linear, high-molecular-weight polymer that is comprised of repeating disaccharide that consist of (β1→3)D-glucuronate-(β1→4)N-acetyl-D- glucosamine. This essential component of the cardiac jelly is synthesized by integral plasma membrane hyaluronic acid synthases. Hyaluornic acid then gets directly exported into the extracellular space to bind salt and water, and also expanding the extracellular compartment (Brown and Papaioannou, 1993; Fenderson et al., 1993).

There has been a wide variety of functions that have been directly and indirectly associated with hyaluronic acid. These functions include cell proliferation, cell adhesion and cell migration. Some of these functions can be directly attributed to the ability of hyaluronic acid to create and fill the extracellular space as well as organizing and also modifying the extracellular matrix. Likewise other rolls of hyaluronic acid can be attributed to its ability to interact and signal through cell surface or cytoplasmic receptors, such as CD44 or RHAMM (Bakkers et al., 2004). 84

Previously, most of the experiments addressing the various functions of hyaluronic acid have been carried out in vitro with a comparably few in vivo analyses reported. To study the role of hyaluronic acid during normal embryogenesis, we generated a hyaluronic acid synthase (Has2) complete knockout mouse. Has2 null embryos have previously been shown to die around E9.5 exhibiting several abnormalities. These include a severely reduced body size and cardiac abnormalities including loss of ventricular trabeculation (Camenisch et al., 2000).

We show here that in vivo, depletion of hyaluronic acid from the cardiac jelly by

HAS2 mutations results in loss of Par complex luminal localization as well as complete loss of ventricular trabeculation. Consequently, this leads to an essentially random orientation of cardiomyocyte spindle orientation and division plane. These findings would suggest that a normal composition of cardiac jelly is therefore required to localize and direct par complex polarity in in vivo cardiomyocytes, leading to the development of ventricular trabeculations.

Hyaluronic acid does not signal myocardial polarity through CD44

What is the mechanism through which hyaluronic acid acts? The complimentary receptor for hyaluronic acid, CD44, activates downstream pathways that converge on

Ras activation, however previous studies have shown that CD44 null mice live and do not exhibit any cardiovascular phenotype (Schmits et al., 1997).

Of importance, we show here that mutations in the hyaluronic acid receptor

CD44 do not affect Par complex localization or polarized cell division. These results 85 argue that HA in the cardiac jelly does not directly control cell polarity but rather may modulate inductive signalling cues. This is analogous to how the ECM protein Fibrillin indirectly modulates TGF-β signalling during mammalian aortic development (Loeys et al., 2013). Likewise, hyaluronic acid may serve as a co-stimulatory molecule and thus acts downstream with various known receptor-mediated signaling pathways.

The role of Prkci during early cardiogenesis

The Par3–Par6–Prkci complex was originally identified as polarity proteins required to establish anterior–posterior polarity of the Caenorhabditis elegans embryo.

It has also been found to be essential for apico-basal polarity of Drosophila and mammalian epithelial cells (Hawkins and Garriga, 1998).

The PKC family of isoforms includes 12 known isoenzymes and has been classified into three large subfamilies. These include the classical PKC isoforms (α, β1,

β2, and γ), the novel PKC isoforms (δ, ϵ, θ, η, and μ), and the atypical PKC isoenzymes (λ,

τ, and ζ). The atypical protein kinases C (aPKC) isoforms ι/λ and ζ have been shown to play crucial roles in many cellular processes including development, cell proliferation, differentiation and cell survival. For the current study, we investigated a possible role for the 74-kDa λ isoform of PKC (Prkci). To study molecular mechanisms of cell polarity formation in the early embryonic mouse heart, we generated a Prkci conditional knockout mouse line in which the Prkci gene is inactivated in early embryonic cardiomyocytes under the control of the Nkx2.5 promoter. 86

We demonstrate that loss of Prkci results in aberrant mitotic spindle alignment, loss of asymmetric cardiomyocyte division, loss of normal myocardial trabeculation.

Previously, a report by Soloff et. al, 2004 described an embryonic lethal phenotype when the Prkci gene was knocked out by conventional gene targeting (Soloff et al.,

2004). Our generated conditional knock out mouse lines for Prkci showed an embryonic lethal phenotype as well.

Although our results demonstrate that Prkci mediated polarized cell division is required for trabecular formation during early cardiogenesis, they do not rule out the possibility that directed cell migration also contributes to this process. In zebra fish, directed cell migration appears to play a central role in trabecular formation (Liu et al.,

2010). However important differences exist between zebrafish and mammals including the highly validated findings showing that cardiomyocyte proliferation occurs in the trabecular myocardium during development (Liu et al., 2010) and regeneration in zebrafish (Jopling et al., 2010), but predominantly in the compact myocardium in mammals (Buikema et al., 2013; Jeter and Cameron, 1971). Furthermore, the developing zebrafish heart is initially constituted from trabecular and embryonic primordial layers, with a third outermost cortical layer created during maturation into adulthood (Gupta and Poss, 2012).

In the skin, epidermal deletion of Prkci results in altered distribution of perpendicular versus planar cell division with an increase in perpendicular division (not planar or oblique divisions) (Niessen et al., 2013). In contrast Prkci deletion in the 87 myocardium results in an increase in planar cell division. In both cases however, the loss of normal polarization results in abnormalities of the cellular differentiation program required for normal morphogenesis.

These findings argue that in the heart, as in multiple other organ systems, the

Par machinery coordinates normal cellular differentiation by facilitating perpendicular cell division and promoting the differentiation of trabecular myocardium from compact myocardium.

In vitro cardiomyocyte polarization

A scratch-induced cell migration in astrocyte monolayers is associated with the spatially restricted activation of a Par3–Par6–Prkci complex. In this case, the localization of the polarity complex at the tip/leading edge of the growing axon in the developing neuron is essential for its polarized axonal growth as well as directional migration and polarization (Etienne-Manneville and Hall, 2003).

We performed a similar assay of migrating embryonic cardiomyocytes. To determine if myocardial cells are capable of polarization during in vitro culture, we performed a scratch-wound assay on cardiomyocytes isolated from E12.5 mouse hearts.

In response to the scratch, embryonic cardiomyocytes at the leading edge reoriented their Golgi apparatus to face the wound. However when we repeated the experiment with GF109203X, a small molecule inhibitor of atypical protein kinase C, this prevented

Golgi reorientation towards the wound edge. This result demonstrates that embryonic cardiomyocytes can polarize in vitro and that this polarization requires Prkci. 88

The role of NuMA during early cardiogeneis

Downstream of Prkci, NuMA along with cytoplasmic dynein, has been previously been shown to contribute to the arrangement of microtubules toward the poles of the mitotic spindle and also directly binding centrosomes to the cell spindle microtubules.

In addition, during cellular interphase, NuMA has been shown to accumulate specifically in the cell’s nucleus where it will participate in aspects of the nuclear structure as well as the nuclear function but then redistributes to the spindle poles early in mitosis.

(Khodjakov et al., 2003; Merdes et al., 1996).

Several reports have shown that NuMA has a large role in organizing as well as stabilizing the mitotic spindle apparatus. For example, when mitotic cultured cells where microinjected with anti-NuMA antibodies, this led to a complete disruption of their mitotic spindles (Kallajoki et al., 1991, 1992). In addition, transfection of a mutant form of NuMA that was unable to bind to the spindle resulted in abnormal spindle morphology with the polar regions losing their normal shape (Compton and Luo, 1995).

Furthermore, complete NuMA depletion eliminates bipolar spindle assembly.

This leads to unusually long microtubules grouped around condensed chromatin

(Merdes et al., 1996). Thus to study the molecular mechanisms of cell polarity formation in the early embryonic mouse heart downstream of Prkci, we generated a NuMA conditional knock-out mouse line in which the NuMA gene is inactivated in early embryonic cardiomyocytes under the control of the Nkx2.5 promoter. 89

Here we demonstrate that a conditional loss of NuMA results in phenomimics the Prkci conditional null mouse embryo by which loss of NuMA leads to aberrant mitotic spindle alignment, loss of asymmetric cardiomyocyte division, loss of normal myocardial trabeculation, and a resulting loss of embryonic viability.

Lineage tracing of early trabeculations

Our results from our knockout experiments are consistent with the hypothesis that if cell polarity is required for myocardial trabeculations to occur, then individual trabeculations are derived from cardiomyocytes undergoing perpendicular cell division.

This therefore suggests that individual trabeculations may be derivative from the same mother cell. To directly test this hypothesis, we have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter system of the R26R-

Confetti 2.1 mouse line wherein Cre-mediated recombination results in one of four

ﬂuorescent markers being randomly placed under control of the constitutive CAGG promoter (Snippert et al., 2010).

We bred the R26R-Confetti mouse line to the Nkx2.5-cre mouse line to examine cell fate mapping of early cardiomyocytes. Two-photon excitation microscopy of E10.5 transgenic hearts revealed that cells of an individual ventricular trabeculation were of the same color, consistent with them being derived from a single mother cell. This provides strong evidence that supports our initial hypothesis that cellular polarity leads downstream to perpendicular/oriented cell divisions. These oriented cell divisions are therefore required for normal myocardial trabeculation and cardiogenesis to occur. 90

Conclusions and perspectives

Collectively our results layout a new molecular mechanism for early cardiac morphogenesis. We demonstrate that in response to inductive signals from a normal composition of extracellular matrix in the cardiac jelly, Prkci and its other partners in the

Par complex signaling pathway orients myocardial cells relative to the inside (lumen) versus the outside of the heart. Furthermore, this polarization directs oriented cell divisions of luminal myocardial cells to driving trabeculation in the early developing heart.

Remarkably we therefore find that building the mammalian heart depends on the same signaling pathways used in worms (which do not even have a heart). These data are relevant to the spatial localization and signaling cues that drive organ morphogenesis and architecture. Our findings are also relevant to cardiac regenerative biology, where the generation/rebuilding of a functional heart requires that the polarity of individual cells be coordinated with the overall organization of the tissue.

Polarity signaling during human disease

A hallmark of tissue development is that the polarity of an individual cell is coupled to the organization of the organ as a whole (Horvitz and Herskowitz, 1992).

Defining the processes whereby cellular polarity is integrated with organogenesis is important not only for understanding normal development, but also for understanding the pathogenesis of human congenital diseases. Aberrant development of myocardial trabeculations for example can result in non-compaction cardiomyopathy (Luxan et al., 91

2013) and has been linked to aberrant signaling from the endocardium to the myocardium.

Recapitulating the normal developmental processes that drive cell polarity and tissue organization will also be important in the regenerative approaches of cardiovascular medicine. The human heart has a limited endogenous regenerative capacity underscoring the need for new therapies.

Understanding myocardial polarity is essential for cardiovascular regenerative medicine

Recent work has opened the possibility for direct reprogramming of endogenous cardiac fibroblasts into functional myocardial cells (Qian et al., 2012; Song et al., 2012). An important challenge however is to not only expand the pool of functional cardiomyocytes, but also to promote their alignment and cellular integration with the native myocardium. Understanding the pathways that direct normal cardiomyocyte polarity in vivo will therefore be instrumental for the successful therapeutic application of cardiac regeneration. 92

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