Cell Polarity in Hematopoietic Stem Cell Quiescence, Signaling and Fate Determination

A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements to the degree of

Doctor of Philosophy (Ph.D.)

in the Department of Cancer and Cell Biology of the College of Medicine March 27, 2020

Mark Jordan Althoff

B.S. Murray State University, 2013

Dissertation Committee:

Jose A. Cancelas, MD, PhD (Chair) Yi Zheng, PhD Marie-Dominique Filippi, PhD Hartmut Geiger, PhD Daniel T. Starczynowski, PhD Richard Q. Lu, PhD

Abstract

Hematopoietic stem cells (HSC) self-renew and differentiate through changes in polarity.

Polarity has been described as a major driver of asymmetric cell division, and in particular, Cdc42 allocation accurately predicts HSC asymmetric division potential. Few proteins responsible for establishing or maintaining cellular polarity (outside of Cdc42) have been investigated among

HSC, and of those that have, many have been deemed functionally dispensable. Scribble is a multi-modular cytoplasmic scaffolding protein that coordinates the spatial organization of cell fate determinants and acts as a molecular hub for a variety of signaling proteins. The contributions of

Scribble on cellular polarity establishment and maintenance in neuronal stem cells and epithelial cells is well characterized, however such mechanisms are yet to be defined in HSC.

We discovered that Scribble controls HSC fate and function by acting as a molecular hub for signaling proteins like the Hippo pathway kinase, Lats1, and the effectors, Yap1 and Taz. The

Hippo pathway controls proliferation and growth of multiple mammalian tissues, yet its role in HSC remains controversial. We found that Yap1 is predominantly polarized in the cytosol of HSC through a Scribble PDZ domain-mediated interaction. Deletion of Yap1 and Taz induces a loss of

HSC quiescence, self-renewal and reconstitution following serial myeloablative 5-fluorouracil treatments, indicating a functional dependency for these effectors. We provide the first functional evidence that Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity, regulating both

HSC quiescence and fate determination in vivo. Deletion of Scribble disrupted Yap1 co- polarization with Cdc42 and decreased Cdc42 activity, resulting in apoptosis of non-self-renewing daughter cells. This data suggests that Scribble/Yap1 co-polarization is indispensable for Cdc42- dependent activity on HSC asymmetric division and fate. The combined genetic loss of Scribble,

Yap1 and Taz in HSC further decreases Cdc42 expression and activity, and is associated with transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, and subsequent

Rac activation and restoration of HSC fitness. Our data indicate that Scribble coordinates the cytosolic functions of Yap1 and Taz with Cdc42 activity and is required for HSC fate determination.

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We also identify a potential novel mechanism by which Scribble coordinates HSC activity in response to stress. Scribble deficient HSC retained cellular quiescence after interferons type I

(IFN-I) stimulation. IFN-I are microenvironment cytokines produced during the physiological response mounted to combat a viral infection. In bone marrow hematopoiesis, IFN-I induce proliferation of HSC. Clinically, patients treated with IFN-I, as well as individuals suffering from

IFN-I associated chronic disease, often exhibit sustained hematological cytopenias and HSC failure. The precise molecular mechanisms that govern HSC behavior in response to IFN-I are still unclear. Our data highlights that the deficiency of Scribble in HSC rendered them insensitive to IFN-I mediated activation. As a result, Scribble deficient HSC treated with IFN-I are functionally more fit, displaying increased competitive reconstitution abilities during serial transplantations. No discernible differences in Stat-1 (the major effector of IFN-I signaling) activity were observed when measuring phosphorylation status, nuclear translocation and transcriptional response within wild- type (Wt) and Scribble deficient HSC following IFN-I exposure. Ly6a transcript levels are appropriately upregulated following IFN-I stimulation, however the encoded stem cell antigen-1

(Sca-1) protein localization was significantly decreased on the membrane surface. These data provide compelling evidence for a role of Scribble in coordinating HSC endosomal membrane trafficking to drive IFN-I mediated HSC activation.

Preface

To address the role of Scribble in HSC polarity and function, I undertook two major research projects. The first project seeks to understand the molecular mechanisms associated with establishing HSC polarity to coordinate stem cell divisions and fate, employing both constitutive and inducible hematopoietic-specific Scribble-deficient animal models, reconstitution assays, and intracellular protein trafficking analysis using structure-function mutants of Scribble. The second project was aimed towards integrating the clinical implications of Scribble-mediated cellular polarity and HSC stress-response to Interferon signaling. As a result, the work presented in this dissertation has been previously published in, or is in preparation for, the following peer-reviewed journals:

1. *Singh A, *Althoff MJ, Cancelas JA. Signaling Pathways Regulating Hematopoietic Stem Cell and Progenitor Aging. 2018. Current Stem Cell Reports. 4(2):166-81. 2. Nayak RC, Hegde, S, Althoff MJ, Wellendorf AM, Mohmoud F, Perentesis J, Reina- Campos M, Reynaud D, Zheng Y, Diaz-Meco MT, Moscat J, Cancelas JA. The signaling axis atypical protein kinase C λ/ι-Satb2 mediates leukemic transformation of B-cell progenitors. 2019. Nat Commun. 10(1): Article Number 46. 3. Althoff MJ, Nayak R, Hegde S, Wellendorf AM, Bohan B, Filippi MD, Xin M, Lu QR, Geiger H, Zheng Y, Diaz-Meco MT, Moscat J, Cancelas JA. Yap1/Scribble polarization is required for hematopoietic stem cell division and fate. Under Review, Blood. 4. Althoff MJ, Wellendorf A, Diaz-Meco MT, Moscat J and Cancelas JA. Scribble mediates IFN-I induced activation of HSC through its regulation of Sca-1 and Akt activity independent of Stat1 effector response. Manuscript in Preparation.

Acknowledgements

First, I would like to take a moment to thank the University of Cincinnati and the Cancer and Cell biology (CCB) graduate program for their continuous guidance and support through my PhD studies.

A large proportion of my gratitude has to go to my PhD mentor, Dr. Cancelas. Without a doubt, Dr. Cancelas has personalized my training and intellectual development to highlight my best attributes, framing me into a productive research scientist. He has an unrivaled devotion for his pupils and a tremendous dedication to health sciences, both attributes I plan to maintain throughout my academic career.

I have to extend my appreciation to the remaining members of my Thesis Committee (Dr. Zheng, Dr. Filippi, Dr. Geiger, Dr. Starczynowski and Dr. Lu) for their expert direction and continual support.

Lastly, I have to thank my growing family, Sara and Isla, for their unwavering support and the confidence they instill in me to follow my dream of becoming an independent research scientist.

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Table of Contents Abstract iii Preface vi Acknowledgements vii Table of Contents viii List of Figures and Tables x

Chapter 1: Hematopoietic Stem Cell Polarity 1

Part 1: Hematopoiesis 1

Hematopoietic Stem Cells 1 HSC self-renewal and multi-lineage differentiation 6 Aberrant, dysregulated and malignant hematopoiesis 11 Transplantation as curative form of treatment 15

Part 2: Polarity and hematopoietic stem cell fate 18

Polarity and Asymmetric Division 18 Asymmetric inheritance of fate determinants and asymmetric cell division in HSC 20 The Scribble Polarity Complex 23

Chapter 2: Hypothesis and Goals 27

Chapter 3: Yap1-Scribble polarization is required for HSC division and fate 28

Abstract 29 Introduction 30 Results 31

The combined activity of the paralogues Yap1 and Taz is necessary for HSC function. 31

HSC Scribble scaffolds cytosolic Yap1 with upstream inhibitory components of the Hippo Signaling pathway. 33

The PDZ domain of Scribble is necessary for Yap1 cytoplasmic polarization while the LRR domain of Scribble is required for active Lats recruitment. 34

Scribble Scaffolds Polarized Yap1 and Activated Cdc42. 35

HSC Scribble deficiency results in enhanced long term self-renewal capacity. 37

Scribble deficiency decreases survival of nascent non self-renewing HSC clones. 38

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Additional deficiency of Scribble restores HSC fitness of Yap1/TazΔ/Δ HSC and associates with Rac activation. 40

Discussion 41 Material and Methods 44 Acknowledgements 52 Author contributions 52 Disclosure of conflicts of interest 52 Figures and legends 53

Chapter 4: Scribble mediates IFN-I induced activation of HSC through its regulation of Sca-1 and Akt activity independent of Stat1 effector response 77

Abstract 78 Introduction 79 Results 81

Scribble deficient HSC are less responsive to IFN-I mediated HSC-activation and exhibit increased competitive repopulation following IFN-I stimulation. 81

Scribble deficient HSC mount an appropriate Stat1 signaling response following IFN-I stimulation. 82

Scribble deficient HSC display lower Akt activity and Sca-1 membrane expression. 83

Discussion 83 Material and Methods 86 Acknowledgements 91 Author contributions 91 Disclosure of conflicts of interest 91 Figures and Legends 92

Chapter 5: Discussion, Implications and Future Directions 95

Chapter 6: Conclusions 103

Chapter 7: Impact and Relevance 104

References 105

List of Figures and Tables

Chapter 1: Hematopoietic Stem Cell Polarity 1

Figure 1.1 Prevailing models of hematopoiesis and HSC lineage commitment. 5

Figure 1.2 Hematopoietic stem cell division modality. 7

Figure 1.3 Scribble binding partners. 26

Chapter 3: Yap1-Scribble polarization is required for HSC division and fate 28

Figure 3.1 Yap1/Taz are necessary for HSC function. 54

Figure 3.2 Scribble scaffolds components of the Hippo pathway in HSC and controls Yap1 cytoplasmic localization. 56

Figure 3.3 Cytoplasmic polarization of Yap1 is restored in ScribbleΔ/Δ HSC/P with expression of Full length Scribble or PDZ containing mutants. 58

Figure 3.4 Scribble scaffolds Yap1 and Cdc42 in the cytoplasm of HSC. 60

Figure 3.5 ScribbleΔ/Δ hematopoietic reconstitution develops a competitive advantage when serially transplanted by maintaining self-renewal divisions. 62

Figure 3.6 Deficiency of Scribble restores HSC fitness of Yap1/TazΔ/Δ HSC and associates with Rac activation. 654

Supplemental Figure 3.1 Constitutively transcriptionally active Yap1 function is dispensable for HSC functional activity. 676

Supplemental Figure 3.2 Scribble deficient HSC lose polarized Yap1 expression in the cytosol. 698

Supplemental Figure 3.3 Scribble binds to and modulates Cdc42 expression and activation. 70

Supplemental Figure 3.4 Scribble deficiency increases repopulation and self-renewal of HSC. 732

Supplemental Figure 3.5 Scribble deficiency decreases quiescence of HSC and modulates fate. 754

Supplemental Figure 3.6 Triple deficiency of Yap, Taz and Scribble identifies a change in the RhoGTPase activation gene transcriptome and Scribble deficiency results in diminished Cdc42 expression. 766

Chapter 4: Scribble mediates IFN-I induced activation of HSC through its regulation of Sca-1 and Akt activity independent of Stat1 effector response 777

Figure 4.1 Scribble deficient HSC are less responsive to IFN-I mediated HSC activation. 922

Figure 4.2 Stat1 activation and transcriptional impact in response to IFN-I remains relatively unchanged. 933

Figure 4.3 Potential mechanism underlying Scribble deficient HSC activation following IFN-I stimulation. 944

Chapter 5: Discussion, Implications and Future Directions 955

Figure 5.1 Proper HSC self-renewal and asymmetric divisions are coordinated by a polarized Scribble/Yap1-Taz/Cdc42 complex. 955

Figure 5.2 HSC that display increased co-polarization between Scribble, Yap1 and Cdc42, harbor greater asymmetric division potential. 999

Figure 5.3 Apolar Scribble protein localization in primitive durable self-renewing HSC. Error! Bookmark not defined.1

Chapter 1: Hematopoietic Stem Cell Polarity

Part 1: Hematopoiesis

Hematopoietic Stem Cells

Hematopoiesis is the well-characterized process by which blood cells are formed. This step-wise process develops from a small population of self-renewing multipotent hematopoietic stem cells (HSC) to an assembly of progenitors with diverse proliferation and differentiation potentials, which subsequently produce functionally distinct mature blood cell populations. Mature blood cells are categorized into erythrocytes (red blood cells), leukocytes (white blood cells) and thrombocytes (platelets), and coordinate specific cellular and physiological properties, such as gas transportation and exchange, defense against pathogens and wound healing (Tanaka and

Goodman, 1972). Cellular functions such as these are highly conserved and are fundamental to all vertebrate life. Over the last few decades, new technologies and model systems have greatly expanded our understanding of HSC biology and hematopoiesis.

In a healthy adult, approximately 2 million blood cells are produced each second (Ogawa,

1993), with continual replenishment of circulating functional immune cells, red blood cells and platelets. This metric imposes an immense pressure or demand for sustained hematopoiesis throughout our lifetime. HSC are a rare population of blood cells that reside in the adult bone marrow (BM) cavity and are responsible for this lifelong production of blood. Therefore, early modeling of hematopoiesis has often been depicted through a functionally organized hierarchical tree, with self-renewing HSC and multipotent progenitor cells positioned at the apex (Morrison et al., 1997; Morrison and Weissman, 1994) (Morrison and Weissman, 1994). These primitive cells are thought to branch out through a series of successive binary and irreversible fate choices, progressing through distinct intermediate progenitor stages that will ultimately give rise to the full repertoire of blood cells (Akashi et al., 2000; Kondo et al., 1997) (Figure 1.1A). The potential for

a single HSC to reconstitute the tissue in its entirety has allowed for the development of transplantation approaches to treat hematologic disorders like primary immunodeficiency, aplastic anemia or myelodysplasia, along with cancers and other non-specific cancer chemotherapy related side effects.

The first in vivo assay defining HSC function was based on the premise of rescued long- term hematopoietic reconstitution of recipients through bone marrow transplantation following lethal irradiation (Jacobson et al., 1951; Till and McCulloch, 1961). In direct contrast to HSC, committed progenitors are defined by the absence of sustained, long-term, reconstitution or self- renewal in recipients, and possess a restricted lineage differentiation capacity (usually bi- or uni- lineage) that fades after the first 3-5 weeks after transplantation (Doulatov et al., 2010; Yang et al., 2005). In 1988, the use of multi-color fluorescence-activated cell sorting (FACS) techniques with monoclonal antibodies subsequently facilitated the purification and isolation of transplantable murine HSC (with full reconstitution capacity) from their less capable progenitor counterparts

(Muller-Sieburg et al., 1986; Spangrude et al., 1988). Proper identification and purification of hematopoietic stem and progenitor cell (HSPC) compartments that are subjected to functional assessment has since become more stringent and refined (Haas et al., 2018). Across laboratories, HSC are immunophenotypicaly defined in the lineage (Lin) negative, cytokine receptor-tyrosine kinase (c-kit)+, stem cell antigen-1 (sca-1)+ (LSK) fraction based on the expression of (i) CD34 and Flk2, (ii) CD105 or (iii) the SLAM markers CD48 and CD150 (Adolfsson et al., 2001; Chen et al., 2002; Kiel et al., 2005; Osawa et al., 1996). Further refinement of these heterogeneous compartments has been recently established with the use of endothelial protein

C receptor (EPCR), CD49b and CD41 surface antigens (Balazs et al., 2006; Benveniste et al.,

2010; Gekas and Graf, 2013), identifying to identify HSC with durable self-renewal potential (LSK,

Flt3-, CD34-, CD48-, CD150+, CD49b- and EPCR+) (Kent et al., 2009). Studies of HSC self- renewal potential that were traditionally monitored through their ability to reconstitute hematopoiesis in lethally irradiated recipient mice over a 4-month time frame, have been extended

to include serial transplantation experiments in order to reveal durable HSC subsets which possess unique lineage commitment potentials (Yamamoto et al., 2013). In accordance with functionally unique lineage-committed (otherwise described as biased) HSC, epigenetic and transcriptional profiling of HSC subpopulations which exhibit specific gene expression patterns that reflect the mature cell population toward which they are biased, highlighting transcriptional heterogeneity between HSC that manifest functionally (Adolfsson et al., 2005; Cabezas-

Wallscheid et al., 2014; Challen et al., 2010).

Similarly, single-cell RNA sequencing and lineage tracing experiments coupled with ex vivo differentiation studies in mice, provided early molecular evidence of heterogeneity and lineage specification within the oligopotent progenitors (Mercier and Scadden, 2015; Notta et al.,

2016; Paul et al., 2015; Perie et al., 2015; Velten et al., 2017). These data imply that the progenitor compartments, previously identified as multipotent or bi-potent (such as granulocyte- monocyte progenitors (GMP) or other bifurcating/ branch point progenitors depicted in Figure

1.1A), actually contain mixtures of lineage-restricted cells harboring unique uni-lineage transcriptional profiles that collectively generate the distinct mature cell populations downstream of their respective branch point. As a result, early and non-binary commitment within progenitors has been depicted as the accumulation of individual lineage-restricted cell populations (Figure

1.1B). Alternatively, hematopoiesis has been presented as a continuum model (Figure 1.1C): In such a model, HSC are influenced through a Waddington-like progression, gradually acquiring lineage-commitment through instruction from the cellular environment, epigenetic landscape, or transcriptional profiles in a continuous manner to increase the probability of one defined lineage

((Macaulay et al., 2016; Nestorowa et al., 2016; Olsson et al., 2016; Pina et al., 2012; Velten et al., 2017). Notably, it is understood within this model of hematopoiesis that the HSC are passing through transitional intermediate states rather than discrete lineage-defined intermediates as were portrayed in the earlier accepted models of hematopoiesis.

Advancement in our abilities to isolate and analyze HSC at the singe cell level has revealed a broad spectrum of molecular, cellular, and functional heterogeneity. This has transformed our perception of how HSC enter lineage commitment to sustain lifelong hematopoiesis. Understanding HSC fate and lineage potential has long been a focal point among stem cell biologists. Within the last few decades, newer models of hematopoiesis have been predicted in an attempt to explain and/or account for the earliest lineage choices in these primitive hematopoietic compartments as well as their subsequent cellular intermediates (Adolfsson et al.,

2005; Akashi et al., 2000; Forsberg et al., 2006; Notta et al., 2016; Perie et al., 2015; Pietras et al., 2015; Yamamoto et al., 2013). A large fraction of such efforts defining lineage fate among

HSC have been performed in the context of ex vivo manipulation and/or hematopoietic transplantation; as a result, the current models of lineage branching are more likely to represent lineage potential rather than their unperturbed native fate. Use of a non-invasive transposable element system has permitted further interrogation into the clonal contribution of HSC in situ

(Rodriguez-Fraticelli et al., 2018; Sun et al., 2014). Tracing the fates of progenitors and HSC in unperturbed hematopoiesis demonstrated that HSC have a limited lympho-erythromyeloid output during steady-state (Busch et al., 2015; Sun et al., 2014) and a fraction of HSC behave as a potent source of megakaryocyte progenitors (MkP), indicating that megakaryocyte fate is a predominant fate choice of HSC in situ (Rodriguez-Fraticelli et al., 2018). In concordance with reconstitution and transplantation, these MkP producing HSC also exhibit the potential for multi- lineage reconstitution following transplantation. These paradigm-shifting lineage tracing experiments highlight the critical differences between studying lineage potential versus native fate in stem cell biology. Collectively, hematopoiesis is maintained by distinct stem and progenitor cell populations in both native and stressed states, adapting to different physiological conditions, indicating that the prevailing views of the hematopoietic hierarchical tree are more flexible than originally presented.

Figure 1.1. Prevailing models of hematopoiesis and HSC lineage commitment. (A) The classic hierarchal model of hematopoiesis: HSC undergo lineage commitment through a stepwise progression of distinct intermediate progenitor stages where lineage decisions are made through subsequent binary branching points (Akashi et al., 2000; Kondo et al., 1997). Notably, a direct pathway into the megakaryocyte/ platelet lineage has been recently reported (Haas et al., 2015;

Notta et al., 2016; Sanjuan-Pla et al., 2013; Yamamoto et al., 2013). (B) Early, non-binary, lineage commitment model: Lineage fate is determined early on and the progenitors are represented as accumulations of individual lineage-restricted cell populations (Mercier and Scadden, 2015; Notta et al., 2016; Paul et al., 2015; Perie et al., 2015; Velten et al., 2017). (C) Hematopoiesis as a continuum: HSC are influenced through a Waddington-like progression and gradually acquire lineage-commitment in a continuous manner, passing through transitional intermediate states rather than discrete lineage defined intermediates ((Macaulay et al., 2016; Nestorowa et al., 2016;

Pina et al., 2012; Velten et al., 2017). Abbreviations used in Figure 1.1: HSC: Hematopoietic stem cell, MPP: Multipotent progenitor, CMP: Common myeloid progenitor, LMPP: Lymphoid biased multipotent progenitor, MEP: Megakaryocyte-erythroid progenitor, GMP: Granulocyte-monocyte progenitor, CLP: Common lymphoid progenitor, Mkp: Megakaryocyte progenitor, EP: Erythroid progenitor, GP: Granulocyte progenitor, MP: Monocyte progenitor, DP: Dendritic cell progenitor.

HSC self-renewal and multi-lineage differentiation

Despite HSC immunophenotypic and functional heterogeneity, a consensus exists that distinguishes and defines a HSC from lesser multipotent progenitors. HSC exhibit two fundamental properties, multi-lineage differentiation potential and maintenance of self-renewal.

Multi-lineage potential refers to the capacity to differentiate and produce all functional mature blood cell lineages, a property shared with many multipotent progenitors high in the hematopoietic hierarchy. However, the second of the two characteristics, self-renewal, grants HSC the specific ability to replicate themselves upon division without noticeable differentiation. HSC fate is coordinated by the precise mode of division they undertake (Figure 1.2). Upon each division, HSC undergo one of the three mutually exclusive cell-division modes: Symmetric self-renewal is the process by which a stem cell division results in two daughter cells and each daughter retains full self-renewal and multi-lineage capabilities; An asymmetric division results with one daughter cell that maintains full stem cell potential while the other is limited with just multi-lineage differentiation abilities; Lastly, a symmetric differentiation or commitment division leaves two daughter cells without self-renewal potential and these cells can only contribute to multi-lineage differentiation.

In order to distinguish these specific division modalities, investigators rely on two major approaches, microscopic imaging of paired daughter cell divisions and functional repopulation/differentiation output. Imaging approaches allow investigators to physically observe

HSC pre- and post-division and define daughter pairs based on fate determinant allocation, the presence or absence of a certain protein or mRNA in divided daughter cells. Fate determinants such as Numb, Myc and CD48 are frequently correlated with differentiation of HSC, while daughter cells expressing the membrane receptor Tie2, maintain stemness and self-renewal capabilities (Cheng et al., 2019; Ito et al., 2012). Alternatively, and perhaps most informative, HSC divisions are monitored and the resulting paired daughter cells are separated and functionally tested with single cell reconstitution and differentiation following transplantation or cultures (Suda et al., 1984; Takano et al., 2004). Here the initial HSC division is defined retrospectively based on

the functional output of each daughter cell. Unfortunately, these two approaches have been mutually exclusive, until very recently, due to the fact that certain culture manipulations such as cellular fixation and permeabilization, are required for immunofluorescent imaging which prevents further downstream functional testing. Novel genetic reporter mouse models are currently being developed that permit live-cell imaging of fate determinants (linked with fluorescent reporters) that allows coupling with the functional analysis of separated daughter cells, ensuring definitive fate determinant and division labeling (Christodoulou et al., 2020; Hinge et al., 2020; Loeffler et al.,

2019).

Figure 1.2 Hematopoietic stem cell division modality. HSC divide through one of three modalities: symmetric self-renewal, ssymmetric division or symmetric commitment. Symmetric self-renewal division results in two daughter cells that each retain full stem cell potential, meaning, the ability to self-renew and maintain multi-lineage differentiation capabilities (Yellow); An asymmetric division results in one daughter cell that maintains full stem cell potential while the other has lost the ability to self-renew and can only contribute to multi-lineage differentiation abilities (Red); A symmetric differentiation or commitment division leaves two daughter cells without self-renewal potential and these cells only contribute to multi-lineage differentiation (Red).

HSC maintain a balance between asymmetric and symmetric cell divisions. This balance between self-renewal and differentiation commitment ensures the appropriate number of stem

cells and differentiated progeny throughout tissue development, homeostasis and repair.

Understanding the mechanisms that govern HSC divisions, which dictate differentiation and self- renewal capacities, remains a central issue in stem cell biology. A large proportion of our understanding of the mechanisms associated with this fate decision is tied to transcription factor biology. Transcription factors that drive programs controlling cellular quiescence, proliferation, self-renewal and differentiation provide an intrinsic element to stem cell divisions. For instance, distinctive and indispensable roles for transcription factors like PU.1 and GATA2 have been discovered for the maintenance of HSC homeostasis (Iwasaki et al., 2005; Rodrigues et al., 2005).

Similarly, specific regulation by transcription factors like cAMP response element-binding (CREB) protein (CBP), p300 and Hoxa9 (homeobox A9) are required for HSC self-renewal (Magnusson et al., 2007; Rebel et al., 2002). In some cases, overexpression of certain Hox genes or Hmga2

(high mobility group AT-hook 2) provides HSC with enhanced self-renewal potential (Copley et al., 2013). In contrast, the enforced expression of the erythroid master gene, Gata1 in HSC results in the exclusive generation of megakaryocyte and erythrocyte lineages, showcasing its transcriptional power on lineage specification and HSC differentiation (Iwasaki et al., 2003).

Transcription factors often function in a complex with chromatin-associated factors.

Chromatin-associated factors, therefore, can modulate the expression or the function of specific

DNA-binding transcription factors and coactivators to coordinate transcriptional programs that are involved in self-renewal and differentiation. Probably the best example of a chromatin associated factor involved in regulating the self-renewal properties of HSC is the polycomb complex protein,

Bmi-1. Bmi-1 is required for the maintenance of self-renewing HSC and determines the proliferative capacity of both normal and leukaemic stem cells (Lessard and Sauvageau, 2003;

Park et al., 2003). Another polycomb complex gene, Ezh2, plays an essential role in preserving

HSC self-renewal potential while preventing premature HSC exhaustion (Kamminga et al., 2006).

Recently, our understanding of HSC maintenance has expanded to incorporate epigenetic regulators and chromatin modifiers that introduce novel heritable mechanisms of stem cell fate.

Chromatin modifiers edit histones by phosphorylation, acetylation, ubiquitylation, SUMOylation and methylation. Together, these modifications alter target gene transcription and control the overall impact of transcription factors. In particular, DNA methylation patterns have been shown to control HSC fate (Broske et al., 2009). The absence of DNA methyltransferase 1 (Dnmt1), a prominent DNA methyltransferase, enhanced cell cycling with inappropriate expression of mature lineage genes, thus facilitating differentiation while compromising HSC self-renewal (Trowbridge et al., 2009). Conversely, methylation patterning from additional DNA methytransferases, like

Dnmt3a and Dnmt3b, seem to be required for proper differentiation (Challen et al., 2014).

Therefore, it is likely that multiple DNA methyltransferases work in concert to create a global methylation landscape that dictates specific transcriptional programs or gene families tailored towards HSC self-renewal or differentiation. Sequencing studies identified mutations involving epigenetic modifiers like DNMT3A, Tet methylcytosine dioxygenase 2 (TET2), and isocitrate dehydrogenase 1 (IDH1) in hematologic malignancies (Ntziachristos et al., 2016). These genes are all directly or indirectly related to DNA methylation and their mutations result in increased HSC self-renewal and even myeloid transformation (Moran-Crusio et al., 2011). Thus, understanding these intrinsic epigenetic and transcriptional networks controlling HSC behavior may provide valuable insight towards eradicating HSC driven hematologic deficiencies and malignancies.

HSC self-renewal and differentiation occur through a cell-autonomous (intrinsic) manner but can also be heavily influenced by instructive environmental signals. Pioneering work with two mast cell deficient mouse strains, the dominant white spotting (W) strain (Russell, 1949), and the steel (Sl) strain (SARVELLA and RUSSELL, 1956), birthed the idea of a ‘stem cell niche’. In other words, an essential microenvironment where HSC reside and receive instructive cues that govern differentiation and self-renewal potential. Through a series of transplantation experiments, in which the donor cells from both W and SI mouse strains were interchanged with respective recipients, conclusions were made that the gene on the W locus was indispensable for functional

HSC while the gene on the Sl locus was critical for an unknown environmental element that was

vital for hematopoiesis but not contained in any hematopoietic cells. The indispensable gene located on the W locus encoded the c-Kit (Nocka et al., 1989; Reith et al., 1990), while the critical gene located on the Sl locus encoded steel factor (SLF), also known as kit ligand or stem cell factor (SCF) (Zsebo et al., 1990). These initial observations followed by careful characterization provided the first evidence that a specific microenvironment or ‘niche’ outside of the hematopoietic system can influence HSC activity. Another essential ligand/receptor pair that was described to be essential for HSC function is thrombopoietin (TPO)/c-Mpl. Genetic elimination of either TPO or c-Mpl leads to a reduction of HSC numbers (Kimura et al., 1998; Solar et al., 1998), suggesting that TPO signaling is a positive regulator of HSC self-renewal. Due to the functional dependencies of HSC for SCF/c-Kit and TPO/c-Mpl signaling, the cytokines SCF and TPO were discovered to be sufficient to support survival and proliferation of purified mouse HSC ex vivo in serum-free cultures (Seita et al., 2007), facilitating ease of HSC perturbation and experimentation.

Apart from classical hematopoietic cytokines, other extrinsic developmental and growth factor signaling pathways have also been shown to be relevant for maintaining adult hematopoiesis. Both Notch and Wnt signaling pathways influence HSC maintenance throughout developmental and adult hematopoiesis (Duncan et al., 2005; Luis et al., 2011). Additionally, HSC expressing the receptor tyrosine kinase, Tie2, adhere to osteoblasts expressing the growth factor angiopoietin (Ang-1) resulting in maintenance of cellular quiescence (Arai et al., 2004). Notably,

Ang-1 treatment in culture (mimicking the niche Ang-1) successfully suppress the proliferation of

HSC and retains long-term repopulating capacity after transplantation (Arai et al., 2004; Zhang et al., 2006). Similarly, the retinoic acid receptor, RARγ, is critical for maintaining the balance between HSC self-renewal and differentiation (Purton et al., 2006). These results suggest that developmental signals and signals from growth factors and morphogens (Ang-1 and retinoic acid) are essential for instructing HSC quiescence and self-renewal abilities.

The diversity of signals coordinating the regulation of HSC self-renewal and differentiation compliment the robustness of such a process, in that a single signaling pathway does not

sufficiently mediate control over HSC fate. Findings such as those mentioned previously are evidence that interactions between the HSC and the niche exist, but more importantly, are required for proper maintenance of the hematopoietic system. To this day, there is much debate concerning the true location and identity of the HSC niche with compelling evidence for a myriad of stromal cell types including endosteal osteoblasts, perivascular mesenchymal stem cells, endothelial arteriole/sinusoid, adipocytes, macrophages and megakaryocyte populations (Pinho and Frenette, 2019). Given that there are numerous reported niche cell populations, each with distinct roles in the regulation of HSC, it is highly likely that HSC heterogeneity could be a direct product of specific niche signaling and instruction. Advanced imaging analysis, coupled with single cell transcriptomics of prospective niche cells (Baccin et al., 2020; Christodoulou et al.,

2020), is bringing us closer to an understanding of the exact molecular, cellular and spatial composition of distinct bone marrow niches, and the respective instructional cues responsible for preserving lifelong hematopoiesis by maintaining HSC differentiation and self-renewal.

Aberrant, dysregulated and malignant hematopoiesis

Perturbation to the natural balance between HSC self-renewal and differentiation through physiological stress and/or oncogenes can increase the likelihood of life-threatening hematologic disorders such as cytopenias and blood cancers, including leukemia, lymphoma and myeloma.

HSC are largely quiescent cells that divide very infrequently, an estimated four to five traceable divisions have been predicted in the cells lifespan before they lose repopulation abilities (Bernitz et al, Cell 2016; Wilson et al, Cell 2008). Each of the different cellular states HSC occupy (i.e., quiescence, proliferation, and differentiation) imposes a unique set of transcriptional demands

(Shyh-Chang et al., 2013). These cellular states of HSC depend on instructional cues from the surrounding marrow microenvironment that activate or inhibit their proliferation (Schuettpelz and

Link, 2013). Apart from the role of HSC in maintaining the homeostasis of blood cell production, they must respond quickly to hematopoietic challenges, such as infection or blood loss, and exit

from quiescence to differentiate and replenish functional blood cells. This remarkable adaptability of HSC, to satisfy the ever-changing demands of various hematopoietic challenges, is critical for survival.

Type I and II interferons (INF), tumor necrosis factor (TNF-α), and lipopolysaccharide

(LPS) directly stimulate HSC proliferation and differentiation, thereby increasing the short-term output of mature myeloid effector leukocytes (Essers et al., 2009; Schuettpelz and Link, 2013).

However, chronic inflammatory cytokine signaling can lead to HSC exhaustion and often contributes to the development of hematopoietic malignancies (Sato et al., 2009). Proteins like

TET2 play a crucial role in myeloid cell function as an epigenetic regulator for cell differentiation, and mediate the transcriptional regulation for inflammatory cytokines such as interleukin 6 (IL6).

During the resolution of inflammation, TET2 normally recruits histone deacetylase 2 (HDAC2) to repress transcription and overall IL-6 levels (Zhang et al., 2015). This epigenetic switch is recognized as an important regulatory step for the termination of the inflammatory state. However, upregulated Il6 expression from HSPC in murine Tet2-knockout mice, in response to acute inflammatory stress, leads to apoptotic resistance via increased expression of pro-survival genes with concomitant decreased expression of pro-apoptotic genes (Cai et al., 2018). Likewise, upon prolonged exposure to TNF-α, HSPC with inactivating TET2 mutations developed a similar resistance to apoptosis with a propensity for myeloid differentiation and disease progression

(Abegunde et al., 2018).

A strong correlation between inflammation, hematologic dysfunction, and disease, exists with age. Adult somatic stem cells play a central role in the homeostasis of tissues where high cellular turnover must be maintained for functionality, like the epidermis, gut epithelium and blood.

These stem cells, along with germinal stem cells, are susceptible to a time-dependent functional decline (Cheng et al., 2008). In the hematopoietic system, aging reduces stem cell function by hindering mobilization, homing, engraftment and lineage commitment, manifesting as a vulnerable immune system and an increased incidence of myeloid malignancies (Bryder et al.,

2006; Liang et al., 2005; Morrison et al., 1996; Rossi et al., 2005; Xing et al., 2006). Hematopoietic cells tend to accumulate somatic mutations over time, but they generally do not impact normal

HSPC function.

Clonal hematopoiesis (CH) describes a state in which a single HSC clone gives rise to a disproportionate number of that individuals mature blood cells. Clonality shifts are considered a novel hallmark of aging in hematopoietic tissue and age-associated disease. Higher levels of clonality have been observed in hematopoietic aging where only a few clones of HSC actively contribute to the production of peripheral blood cells (Busch et al., 2015; Sun et al., 2014;

Verovskaya et al., 2013). Several groups have identified somatic mutations in the blood cells of healthy older adults using high-throughput targeted sequencing methodologies on large data sets

(Genovese et al., 2014; Jaiswal et al., 2014; McKerrell et al., 2015). Certain rare somatic mutations can be detected in younger individuals; however they increase in frequency with age, reaching approximately 10-20% of the clonal contribution (Genovese et al., 2014; Jaiswal et al.,

2014). Notably, an equivalent state of clonality is also prevalent in the epithelium of skin

(Martincorena et al., 2015) and the esophagus (Martincorena et al., 2018), suggesting that somatic mutation-driven clonal expansions may be a characteristic of aging among several tissue specific stem cell populations.

CH in these older adults has been largely associated with mutations in a DNMT3a, TET2 and a polycomb group protein transcriptional repressor (ASXL1), which implies a causal relation for these genes in aging-associated clonality. Interestingly, DNMT3a, TET2 and ASXL1 encode proteins that epigenetically regulate transcription, suggesting that epigenetic clonality increases with aging, where clones of certain epigenetic profiles are preferentially selected for and maintained by the aging microenvironment. Among the most frequently mutated genes in acute ayeloid leukemias (AML) are DNM3a, TET2 and ASXL1, which renders these genes of high importance. Leukemias are an extreme case of clonal hematopoiesis, yet we cannot ignore the

striking connection between somatic mutations during clonal hematopoiesis and hematopoietic malignancy.

To clinically bridge the gap, clonal hematopoiesis of indeterminate potential (CHIP) has been described, and is a risk factor for hematopoietic neoplasia, in which somatic mutations are found in the cells of the blood or bone marrow, but no other criteria for neoplasia are met

(Steensma et al., 2015). The minimum variant allele frequency (VAF) for genetic variants in individuals to meet the criteria for CHIP is ≥2%(Steensma et al., 2015). Furthermore, CHIP is associated with and is often a potential precursor for myeloid diseases. In fact, the annual risk of transformation into a hematologic neoplasm is 0.5-1% however, its prevalence rises with age.

Current pre-neoplastic states, including CHIP, idiopathic cytopenia of undetermined significance

(ICUS), and clonal cytopenias of undetermined significance (CCUS), are classified primarily based on the presence of clonality, and on the presence of peripheral cytopenia (Bejar, 2017;

Steensma et al., 2015). Myeloid malignancies occur when individual HSPC mutant clones proliferate, expand and skew towards myeloid lineages. These include myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and AML. The World Health

Organization describes MDS as having ineffective hematopoiesis, characterized by abnormal hematopoietic cell shapes (morphological dysplasia) and peripheral cytopenia (Arber et al., 2016).

MPN are characterized by the proliferation of mature myeloid cells, usually lacking morphological dysplasia (Vardiman et al., 2009). AML involves the proliferation of immature myeloid cells

(“blasts”) related to mutations that block normal HSPC differentiation and, like MPN, can involve recurrent mutations associated with cellular proliferation (Arber et al., 2016; Vardiman et al.,

2009). HSC and their progenitor descendants are often the source for a range of hematologic malignancies. Thus, the pre-malignant and malignant progeny would ostensibly retain residual molecular features from the cell of origin. HSC heterogeneity then becomes extremely relevant in this context, both in understanding the molecular underpinnings by which diseases such as

myelodysplasia and leukemia evolve, and in comprehending the phenomenon of resistance to therapy which leads to disease relapse.

Transplantation as curative form of treatment

HSC represent the therapeutic component for successful bone marrow transplants.

Successful transplantation is made possible by the remarkable ability of stem cells to home and engraft to the appropriate niche and provide long-term, full lineage reconstitution (Aizawa and

Tavassoli, 1988; Boggs, 1984; Champion et al., 1986; Hardy and Tavassoli, 1988; Lepault and

Weissman, 1981; Vos et al., 1980). Allogeneic HSC transplantation (HSCT) has become a clinical standard, and often the sole curative approach, to treat genetic hematologic diseases such as primary immune deficiencies, hemoglobinopathies, storage and metabolic disorders, congenital cytopenias and malignancies (Boelens et al., 2013; Walters, 2015). HSC are harvested from healthy donor bone marrow, peripheral blood, or umbilical cord blood. Most procedures require the cells from the donor to be human leukocyte antigen (HLA)-matched with the patient recipient.

Though HSC have been a viable therapeutic for decades, many patients lack a HLA-matched donor (Hatzimichael and Tuthill, 2010). In certain situations, the patient’s own HSC may be harvested and banked to ensure matching for later therapeutic use, however this may become quite costly. Regardless, reduced HLA-matching between the recipient and the donor increases the risks of graft rejection and graft versus host disease (GVHD). Rejection of an HSC graft commonly leaves the patient in a compromised state, resulting in an urgent need to restore hematopoiesis in order to prevent complications from pancytopenia and infection. Thus, GVHD is a major cause of transplantation morbidity, and even mortality. In many cases, GVHD can impose a chronic inflammatory state (Billingham et al., 1959; Cooke et al., 2017). As a result, immediately preceding and following HSCT, high levels of immune suppression are necessary to dampen the immunological risks, but these modifications often contribute towards morbidity. Methods to

reduce GVHD in allogeneic HSCT have been significantly improved by the removal of selective T cell populations (i.e. T cell receptor α/β depletion or naive T cell depletion), and by the use of post- transplantation cyclophosphamide (Fuchs, 2015; Muccio et al., 2016). Nonetheless, the lack of suitable HLA-matched donors and the potential immune complications present significant clinical barriers to successful allogeneic HSCT.

Since HSCT is a curative approach in many immunohematological diseases, HSCT represents the cornerstone of ex vivo gene therapy. Genetically modified HSC for HSCT should provide complete avoidance of the major immunological complications associated with HSCT, and improve the outcomes for patients with genetic hematologic disorders. Indeed, current approaches to gene therapy using lentiviral vectors have produced clinical benefits for a variety of genetic hematopoietic diseases, and consistently achieve stable frequencies of gene-corrected blood cells (of all lineages), indicating successful engraftment and long-term generative capacity of the gene-modified HSC (Cartier et al., 2012; Enssle et al., 2010);(Aiuti et al., 2013; Biffi et al.,

2013). Recent developments in gene editing have led to investigations into its application for ex vivo gene correction in HSC (Wright et al., 2016). Despite this progress, a common struggle in the efforts to improve HSCT is that ex vivo manipulation of HSC often alters stem cell potential, rendering them less functional for durable repopulation. Therefore, a search of molecular targets to expand functional HSC remains a priority, particularly when manipulation of cellular quiescence and fate are required. Understanding the mechanisms by which HSC maintain quiescence and initiate self-renewal would improve the translational impact of current ex vivo gene manipulation/ transplantation approaches.

The introduction of induced pluripotent stem cells (iPSC) technology has made it possible to derive pluripotent stem cells from the patient’s own tissues, thus creating a viable source of autologous HSC for individual patients in need of a transplant (Takahashi et al., 2007). Pluripotent stem cells are, in theory, capable of differentiating into all cells that make up an organism, including HSC. Thus, de novo generation of HSC from human pluripotent stem cells represents

a high priority for HSC biology and regenerative medicine. Early efforts provided evidence that human CD34+ CD45+ cells have been successfully isolated from teratomas (tumors comprised of multiple tissue types) that had formed following subcutaneous injection of human iPSC into immunodeficient mice (Amabile et al., 2013; Suzuki et al., 2013). As proof-of-principal, such hematopoietic progenitors were able to successfully engraft and reconstitute hematopoietic tissue during serial transplantation experiments, however significant improvement of engraftment and extensive functional analysis are still necessary before potential clinical use. These reports imply that functional human HSC can in fact be successfully derived from human pluripotent stem cells sources with the appropriate experimental manipulation, however, despite significant advancements, efficient derivation of functional HSC with the capability for definitive in vivo engraftment and multi-lineage potential remains a challenge.

Part 2: Polarity and Hematopoietic Stem Cell Fate

Polarity and asymmetric division

Cell polarity refers to the spatial differences in shape, structure, and function within a cell.

In its most simplistic form, cellular polarity is analogous to cellular asymmetry, i.e. the differential partitioning or segregation of cellular contents within a cell that enables them to carry out specialized functions. Examples of asymmetrically distributed cellular contents include: cytoskeletal components, organelles and macromolecules like proteins, lipids or RNA determinants. Cell polarity is one of the most basic properties of all living cells and its dysregulation has been considered to be a hallmark of cancer due to its contribution to epithelial cell boundaries and tissue architecture and the regulation of a process known as epithelial to mesenchymal transition (EMT) (Vaira et al., 2011). In addition to EMT, a major step required for solid tumor invasion and metastasis, cellular polarity controls a variety of cellular processes, including spatially restricted signaling axis, reorganization of the cytoskeleton, membrane/ vesicle trafficking, and cellular divisions.

An asymmetric cell division is defined as a division that generates two daughter cells that have functionally different fates, which can be characterized by differences in size, morphology, or gene expression patterns, between the two daughter cells (Horvitz and Herskowitz, 1992).

Asymmetric cell divisions largely occur as a result of simple intrinsic asymmetries of fate determinants within the dividing cell. Alternatively, and less frequently, cells can undergo a niche- mediated asymmetric division whereby the cell specifically orients the division plane so that only one cell of the two daughter pair remains in contact with the surrounding niche, thus preserving stem cell identity. The first asymmetrically inherited protein determinant to be molecularly and functionally characterized, Numb, provided direct evidence for the intrinsic mechanism of asymmetric division (Rhyu et al., 1994). Numb, is a gene observed to be critical in the determination of cell fate during neuronal precursor divisions in developing Drosophila embryos,

as mutants defective in the numb gene lack most of the neurons of the embryonic peripheral nervous system (Uemura et al., 1989). Indeed, asymmetric distribution of numb protein was observed during sensory organ precursor cell division and confers distinct fates to daughter cells

(Rhyu et al., 1994). Thus, the differential distribution or inheritance of numb in neuronal precursors generates an asymmetric division in which the daughter cells acquire distinct identities (Rhyu et al., 1994). Studies of the development of Drosophila nervous system have led to the identification of a myriad of genes that function in asymmetric cell division, such as, Prospero, Miranda, Staufen and Inscuteable (Doe et al., 1991; Kraut et al., 1996; Li et al., 1997; Shen et al., 1997).

Similarly, asymmetric cellular divisions have been extensively studied in Caenorhabditis elegans, in particular, the first several cellular division of the developing zygote. Five asymmetric divisions produce six founder cells that are absolutely critical for establishing the principal axes

(anterior-posterior, dorso-ventral and lateral/ left-right axes) and diversity during C. elegans development (Sulston et al., 1983). Several key proteins which are responsible for setting up C. elegans zygote polarity were identified in a pioneering genetic screen (Kemphues et al., 1988), these included the so-called par complex genes (partitioning defective), par-1-6. Mutations in these particular genes resulted in defective anterior-posterior polarity establishment at the one- cell stage, as evidenced by the generation of daughter cells with altered size, spindle orientation, cell cycle progression and fate. Pkc-3, a seventh member of this group, was identified later by homology to an atypical protein kinase C (aPKC) (Tabuse et al., 1998). PAR-3 and PAR-6 form a complex with aPKC and the distribution of this complex is restricted to the anterior cortex during the end of prophase. PAR-2 and PAR-1 localize in a reciprocal manner to the posterior cortex.

Despite their functional similarity, Par-proteins are quite divergent in sequence homology: Par-1,

Par-4 and aPKC are serine/threonine (Ser/Thr-kinases), Par-3 and Par-6 are PDZ (PSD-95-Dlg-

ZO-1) domain proteins, Par-2 contains a RING-finger domain, and Par-5 is a member of the 14-

3-3 class of proteins. Their homologs are directly involved in the establishment of the Drosophila

anterior-posterior body axis (Macara, 2004) and were described to regulate epithelial and neuronal polarity in Drosophila and higher vertebrates (Wodarz et al., 1999).

Collectively, cellular polarity plays a significant role as a driver of asymmetric division throughout development and evolution. However, the precise molecular mechanisms by which these polarity proteins function seem to be tissue specific, context dependent and even, in some cases, vastly different from their invertebrate origins. As a result, this overly simplistic interpretation of cellular polarity mediated division, particularly in HSC, has been a focal point of investigation in stem cell biology.

Asymmetric inheritance of fate determinants and asymmetric cell division in HSC

Polarity is largely associated with specialized and compartmentalized functions in HSC, like homing, migration, adhesion, endosomal trafficking, proliferation and division. We first learned that C. elegans and Drosophila development relies heavily on asymmetric cell divisions (Rhyu et al., 1994; Sulston et al., 1983). Later, asymmetric segregation of functional cell fate determinants has proven to play a large role in stem cell fate. An accumulating body of evidence focusing on the mechanisms that control cellular asymmetry and division among eukaryotic single-celled microorganisms, Saccharomyces cerevisiae or budding yeast, links polarity directly with asymmetric division and the overall stem cell aging process (Aguilaniu et al., 2003; Erjavec et al.,

2007; Liu et al., 2010; Shcheprova et al., 2008). Specifically, these reports suggest that polarity likely evolved as a mechanism to avoid clonal senescence by establishing an aging mother cell that accumulates oxidatively damaged and/or aggregated proteins, while a rejuvenated daughter cell inherits limited amounts of toxic and deteriorated material, thus preserving the fitness of the other daughter cell, akin to HSC self-renewal. Precluding the inevitable time-dependent functional decline of somatic stem cells, aged HSC show reduced asymmetry with respect to established polarity markers like tubulin, nuclear histone H4 lysine 16 acetylation and Cdc42 (Florian et al.,

2012; Kohler et al., 2009), suggesting that polarity may contribute to the mechanisms controlling hematopoietic stem cell division and fate throughout our lifespan.

A polarized dividing cell has a differential partitioning of protein or RNA determinants into the two daughter cells prior to mitosis. The resulting polarization or asymmetric distribution of cytoskeletal proteins and other cellular constituents within the dividing cells often dictates binary cellular fate decisions. Indeed, asymmetric segregation of molecular fate determinants has been proposed as a potential mechanism for asymmetric stem cell divisions among both murine and human hematopoietic stem and progenitor cells (Beckmann et al., 2007; Brummendorf et al.,

1998; Cheng et al., 2019; Ito et al., 2012; Wu et al., 2007). HSC frequently display an asymmetric distribution in the proliferation and fate determinant Cdc42 (a small Rho-guanosine triphosphatase (GTPase) (Florian et al., 2012; Florian et al., 2013). Perhaps the most direct evidence connecting cellular polarity to HSC function comes from work by Florian et al. in which they show that Cdc42 is polarized alongside tubulin in young HSC. Aged HSC lose Cdc42 polarization and exhibit an increase in Cdc42 activity (Florian et al., 2012). The numbers of apolar

(non-polar) HSC correlate with the observed age-associated decline in HSC function.

Pharmacological intervention using a Cdc42 inhibitor (Casin) can rejuvenate aged HSC to be functionally equivalent to young HSC, informing us that HSC polarity and function are reversible

(Florian et al., 2012). Further investigation into Cdc42 polarity revealed that a shift from canonical to non-canonical Wnt signaling leads to apolar HSC which is responsible for the age-related deterioration in the hematopoietic system. The levels of Wnt5a are significantly elevated in aged

HSC, while expression of canonical Wnt ligands remains unaltered during aging. Remarkably, reduction of the non-canonical Wnt5a signaling in old HSC successfully rejuvenated chronologically aged HSC and restored polar localization of Cdc42 (Florian et al., 2013).

Additionally, Cdc42 hyperactivity (achieved using hematopoietic-specific Cdc42 GTPase- activating protein (GAP)-/- murine models) hindered HSC survival, adhesion and engraftment, while Cdc42 deficient models lead to an increased proliferative state and subsequent exhaustion

of HSPC (Wang et al., 2007; Yang et al., 2007). Collectively, dysregulated Cdc42 polarity is associated with altered HSC self-renewal and differentiation potentials (Florian et al., 2012;

Florian et al., 2013; Wang et al., 2007; Yang et al., 2007). These data indicate that Cdc42 spatial distribution and activity must be tightly regulated for proper HSC function and hint of a crucial role for polarity in stem cell maintenance.

As polarity is a major driver of asymmetric cell division, Cdc42 allocation accurately predicts HSC potential (Florian et al., 2018). Very few polarity proteins, outside of Cdc42, have had such a convincing functional role in HSC (Wu et al., 2007; Zimdahl et al., 2014). Numb, arguably one of the most well-known cell fate determinants throughout development, is indeed polarized in HSC (Loeffler et al., 2019; Ting et al., 2012). aPKC, of the Par complex, mediates phosphorylation and asymmetric membrane localization of Numb (Smith et al., 2007) which presumably, in this context, should define HSC self-renewal and differentiation potential through symmetric and asymmetric divisions. Pharmacological attenuation of aPKCζ signaling induces mobilization and differentiation of hematopoietic progenitors (Petit et al., 2005). Likewise, a shRNA-based screening has suggested that aPKCζ and Par6 could positively regulate mammalian HSC activity (Hope et al., 2010). However, recent evidence from our group has shown that, in contrast to the accepted paradigms, the activity of adult HSC do not depend on either of the Par complex kinase isoforms (aPKCζ or aPKCλ) in vivo (Sengupta and Cancelas, 2011).

Given that aPKC is a core kinase within the cell polarity network (Etienne-Manneville and Hall,

2001, 2003), this discovery may indicate the existence of an alternative signaling network defining

Numb asymmetry in HSC. Regardless, Numb is described to be asymmetrically inherited during

HSC divisions by some (Loeffler et al., 2019; Wu et al., 2007; Zimdahl et al., 2014) while being contradicted by others (Ting et al., 2012). The observation that HSC isolated from mice lacking

Numb and the Numb-like protein (Wilson et al., 2007), Notch1 (Mancini et al., 2005) or the upstream polarity complex genes aPKCλζ (Sengupta and Cancelas, 2011) behave normally, supports the notion that Numb asymmetries are unlikely to functionally contribute to HSC

divisional fate. In addition to Numb, other fate determinants such as, AP2A, Tie2, CD63, Myc, active mitochondria and lysosomal machinery (Beckmann et al., 2007; Cheng et al., 2019; Hinge et al., 2020; Ito et al., 2012; Loeffler et al., 2019; Ting et al., 2012; Vannini et al., 2019; Wu et al.,

2007; Zimdahl et al., 2014), have been suggested to be asymmetrically inherited during HSC divisions. Still, no functional relevance for such asymmetrically inherited fate determinants has been demonstrated in HSC, with the exception of mitochondrial inheritance.

Elegant work performed by Zimdahl et al. identified a cytoplasmic dynein-binding protein, lissencephaly-1 (encoded by Lis1), as a key regulator of HSC fate. Cell polarization through Numb occurred normally in the absence of Lis1, while mitotic spindles failed to orientate perpendicular to the polarization axis, preventing equal segregation of fate determinants and subsequent symmetric division, which led to increased HSC differentiation and depletion of the stem cell pool

(Zimdahl et al., 2014). This result highlights that the ability of HSC to symmetrically divide is an actively regulated process in Numb polarized cells, executed via precise mitotic spindle orientation, and establishes a role for Lis1 in instructing the transition from asymmetric to symmetric cell division in healthy and malignant hematopoiesis. Nevertheless, Lis1 does not influence the initial polarization of fate determinants in HSC prior to division, only the cleavage plane. Although there is compelling evidence for polarity and asymmetric distribution of cellular factors, it remains to be determined, mechanistically, how polarization is established in HSC and what the direct consequences of asymmetric inheritance in daughter cells may be on HSC fate.

The Scribble Polarity Complex

Searching for functionally relevant HSC fate determinants outside the aPKC-numb axis, we analyzed the role of the Scribble polarity complex. The Scribble complex, consisting of Lethal giant larvae (Lgl), Discs large (Dlg) and Scribble, is one of three major polarity complexes that are evolutionarily conserved throughout metazoan phylogeny and coordinates the spatial organization of intracellular proteins. Conditional deletion of Lgl1 in HSC leads to an expansion

in the HSC population during steady-state hematopoiesis with an acquired increase in repopulation capacity, as well as a competitive advantage during serial transplantations (Heidel et al., 2013). Contradictorily, a deficiency of Scribble, its single functional homolog, has been recently reported to have decreased competitive reconstitution ability in an interferon mediated hematopoietic inducible Mx1Cre model (Mohr et al., 2018). Despite the seemingly antagonistic roles that these complex partners might play in hematopoiesis (and hematopoietic stem cell biology), it remains unclear, mechanistically, how these opposing functional effects are mediated.

Both the Scribble and Lgl1 complex partners lack any known intrinsic enzymatic activity, however they contain a number of well characterized protein-protein interacting domains that enable them to bind signaling node proteins (Bonello and Peifer, 2019; Stephens et al., 2018). Based on this premise, it is quite possible that Scribble function in HSC activity does not exclusively depend on

Lgl1, but on binding to other molecular partners.

Scribble belongs to the LAP (LRR and PDZ) protein family (Bilder et al., 2000; Bilder and

Perrimon, 2000; Santoni et al., 2002) with its 16 Leucine-rich repeats (LRR), two LAP-specific domains (LAPSD) and four PDZ protein interacting domains (Figure 1.3). Its LRR domains function to control cell shape and size by tethering proteins to the plasma membrane, as well as by mediating protein interactions like the binding between Scribble and its complex partner Lgl

(Bilder et al., 2000; Bilder and Perrimon, 2000; Santoni et al., 2002) (Figure 1.3). Additionally, the

PDZ domains contribute to the promiscuity of the protein by mediating numerous protein-protein interactions. The PDZ family is one of the largest domain families in the human proteome, containing more than 400 members that typically interact with C-terminal peptides (Nourry et al.,

2003). Consistent with the tumor suppressive origins of the Scribble complex, (Gateff and

Schneiderman, 1969) Scribble has been described as a negative regulator of proliferation in several cell types. The mechanism of cell cycle inhibition elicited by Scribble seems to be related to its ability to interact with known cell cycle regulators like Cdc42/β-Pix (Ivarsson et al., 2014;

Lim et al., 2017), Erk (Nagasaka et al., 2010), APC/β-catenin (Ivarsson et al., 2014), Phosphatase

and tensin homolog (Pten) (Feigin et al., 2014), Phlpp1 (Li et al., 2011) and transcriptional coactivator with a PDZ-binding domain (Taz) (Cordenonsi et al., 2011) (Figure 1.3). Class I PDZ domains recognize the X-S/T-X-ØCOOH motif (X: any amino acid residue, Ø: any hydrophobic residue) and account for the vast majority of Scribble interactions (Stephens et al., 2018).

Scribble localization has recently been reported to be regulated by S-palmitoylation at conserved cysteine residues. Palmitoylation-deficient mutants of Scribble are mislocalized, leading to the disruption of cell polarity and loss of their tumor suppressive activities to oncogenic

Yes-associated protein (YAP), mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways

(Chen et al., 2016). An accumulating body of evidence has shown that apical-basal polarity proteins may regulate the Hippo signaling pathway (Enomoto and Igaki, 2011; Llado et al., 2015;

Mohseni et al., 2014) and consequently the downstream transcriptional co-activators Yap1 and

Taz (Dupont et al., 2011; Huang et al., 2005; Varelas et al., 2010; Wu et al., 2003). Yap1 and Taz contain a class I PDZ binding motif (LTWL-coo-) at their c-terminus through which they have been predicted to bind with Scribble (Sundell et al., 2018), suggesting that successful Hippo signaling is dependent on the expression and scaffolding ability of Scribble.

Yap1 signaling has recently been shown to instruct primitive HSC specification, production, and maturation in vivo (Lundin et al., 2020). Yap1 and Taz have been reported to be expressed in adult HSPC, however their function has been deemed dispensable as a result of loss-of-function, competitive repopulation assays and hematopoiesis output analysis of transgenic animals expressing the active form of the transcriptional coactivator Yap1 (Yap1

S112A) (Donato et al., 2018; Jansson and Larsson, 2012). In this dissertation, we would like to provide evidence that the cytoplasmic functions of Yap1 are more influential to HSC function rather than its transcriptional impact. We have identified that the polarization of Scribble in HSC coordinates the organization and activation of the Hippo signaling pathway via PDZ domain mediated interactions, and it associates with Cdc42 to modulate its activity and regulate HSC divisional fate.

Figure 1.3. Scribble binding partners. Scribble is a multi-modular cytoplasmic scaffolding protein that binds, directly (domain-mediated) or indirectly (co-immunoprecipitation with Scribble but formal biochemical evidence of direct interaction is yet to be shown) with numerous proteins.

Many of these interactions include binding with structural proteins to control processes like cell adhesion, endosomal trafficking and cellular rigidity, while Scribble also acts as a molecular scaffold, facilitating various signaling hubs to regulate Wnt/β-catenin, BMP/TGF-β, PI3K/Akt,

RTK/Ras/MAPK, JNK/p38 and Hippo signaling pathways. In addition, recent reports indicate interactions with known members of the RhoGTPase family and several viral proteins. LAP- specific domain has no know interactions. * indicates interactions between Scribble and its complex partners, Lgl and Dlg (Dlg interaction is mediated through GUKh).

Chapter 2: Hypothesis and Goals

Hypothesis: Scribble coordinates the spatial distribution and activity of crucial signaling pathways to establish HSC polarity and control fate decisions during both normal and stressed hematopoiesis.

Two major project goals were defined to guide our studies and elucidate the precise mechanisms in which Scribble mediates cellular polarity and controls HSC fate:

Goal 1: Determine the role and downstream signaling components with which Scribble interacts to establish HSC polarity and fate decisions.

Goal 2: Elucidate the Scribble-dependent mechanisms that regulate HSC activation and proliferation in response to IFN-I.

Successful identification of the precise molecular mechanisms underlying Scribble mediated HSC polarity, and its ensuing effect on HSC fate, would greatly enhance our understanding of the overall mechanisms that collectively control HSC maintenance and self-renewal. Such knowledge would prove valuable as we strive to identify novel molecular targets for ex vivo HSC gene therapy techniques in order to improve current stem cell transplantation efficacy and ameliorate HSC diseases.

Chapter 3: Yap1-Scribble polarization is required for HSC division and fate

Mark J. Althoff1,2,3, Ramesh C. Nayak1,2, Shailaja Hegde1,2, Ashley M. Wellendorf1, Breanna Bohan2, Marie-Dominique Filippi1,3, Mei Xin1, Q. Richard Lu1, Hartmut Geiger1, Yi Zheng1, Maria T. Diaz-Meco4, Jorge Moscat4, Jose A. Cancelas1,2,3

1Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 2Hoxworth Blood Center, University of Cincinnati Academic Health Center, Cincinnati, OH 3Cancer & Cell Biology Program, University of Cincinnati College of Medicine, Cincinnati, OH 4Sanford-Burnham-Prebys Discovery Cancer Institute, La Jolla, CA

Manuscript is under review with Blood.

Abstract

Hematopoietic stem cells (HSC) enter cell cycle in response to extrinsic cues and self- renew or differentiate through changes in their polarity. Few proteins responsible for establishing or maintaining cellular polarity have been investigated among HSC, and of those that have, many were deemed functionally dispensable. Scribble is a multi-modular cytoplasmic scaffolding protein that coordinates the spatial organization of cell fate determinants and acts as a molecular hub for a variety of signaling proteins, like the Hippo pathway kinase, Lats1, and the effectors, Yap1 and

Taz. The Hippo pathway controls proliferation and growth of multiple mammalian tissues yet its role in HSC remains controversial. We found that Yap1 is predominantly polarized in the cytosol of HSC through a Scribble PDZ domain-mediated interaction. Deletion of Yap1 and Taz induces a loss of HSC quiescence, self-renewal and reconstitution following serial myeloablative 5- fluorouracil treatments, indicating a functional dependency for these effectors. We provide the first functional evidence that Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity, regulating both HSC quiescence and fate determination in vivo. Deletion of Scribble, which again induced a loss of HSC quiescence, disrupted Yap1 co-polarization with Cdc42 and decreased

Cdc42 activity, resulting in apoptosis of non-self-renewing daughter cells. This data suggests that

Scribble/Yap1 co-polarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined genetic loss of Scribble, Yap1 and Taz in HSC further decreases

Cdc42 expression and activity, and associates with transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, subsequent Rac activation and restoration of HSC fitness.

Our data indicate that Scribble is required for HSC self-renewal and links the cytosolic functions of the Hippo signaling cascade effectors with Cdc42 activity in HSC fate determination.

Introduction

The search of molecular targets to expand functional hematopoietic stem cells (HSC) for translational application remains paramount. HSC will respond to microenvironmental cues and self-renew or differentiate according to changes in cellular polarity. HSC frequently display an asymmetric distribution of the proliferation and fate determinant Cdc42 (Florian et al., 2012;

Florian et al., 2013). Cdc42 activity must be tightly regulated for proper HSC function (Wang et al., 2007; Yang et al., 2007). Very few polarity proteins, outside of Cdc42, have such a convincing functional role in HSC (Hinge et al., 2017; Wu et al., 2007; Zimdahl et al., 2014) (Ting et al., 2012).

The observation that HSC isolated from mice lacking Numb and Numb-like (Wilson et al., 2007), upstream Notch1 (Mancini et al., 2005) or aPKC (Sengupta and Cancelas, 2011) behave normally have proven that Numb asymmetries are unlikely to control HSC divisional fate. Searching for functionally relevant HSC fate determinants, we analyzed the role of the Scribble polarity complex.

The Scribble Complex, consisting of Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble, is one of three major evolutionarily conserved polarity complexes that coordinates the spatial organization of intracellular proteins. Conditional deletion of Lgl1 in HSC leads to an expansion in the HSC population with an acquired increase in competitive repopulation capacity (Heidel et al., 2013). On the contrary, deficiency of Scribble has been reported to result in decreased competitive reconstitution ability (Mohr et al., 2018). The mechanism for which the seemingly antagonistic activities of Lgl1 and Scribble in HSC activity is unknown. Both, Scribble and Lgl1, complex partners lack any known intrinsic enzymatic activity, however they contain a number of well characterized protein-protein interacting domains that enable them to bind signaling node proteins (Bonello and Peifer, 2019; Stephens et al., 2018). An accumulating body of evidence has shown that apical–basal polarity proteins may regulate the Hippo signaling pathway (Enomoto and Igaki, 2011; Llado et al., 2015; Mohseni et al., 2014) and consequently the downstream transcriptional co-activators Yes-associated protein 1 (YAP1) and transcriptional coactivator with

a PDZ-binding domain (TAZ) (Dupont et al., 2011; Huang et al., 2005; Varelas et al., 2010; Wu et al., 2003).

Yap1 is believed to play a role in mammalian hematopoietic specification (Goode et al.,

2016)and in cord blood-derived human HSC, Yap1 and its obligatory nuclear partner TEAD1 regulate differentiation of B-cell progenitors (Laurenti et al., 2013). However, formal demonstration of the role of Yap1 and Taz in adult HSC activity is lacking (Donato et al., 2018; Jansson and

Larsson, 2012). Thus, we wanted to explore the concept of Yap1/Taz interacting with and coordinating polarity fate determinants in HSC. Indeed, polarized Scribble coordinates the organization and activation of the Hippo signaling pathway resulting with cytosolic Yap1. We provide the first functional evidence that Scribble and Yap1 coordinate to control Cdc42 location while positively regulating its activity, driving HSC quiescence and fate decisions in vivo. Our data indicates that the Scribble/Yap1 co-polarization is indispensable for Cdc42-dependent activity on

HSC asymmetric division and fate.

Results

The combined activity of the paralogues Yap1 and Taz is necessary for HSC function

In determining whether Yap1 and Taz control hematopoiesis, we first observed that the complete loss of Yap1/Taz results in non-viable pups, whereas the incomplete loss of one to three alleles did not impair the expected Mendelian inheritance ratios from hematopoietic-specific

Vav1CreTg/-;Yap1f/f;Tazf/f mice (Supplemental Figure 3.1A). These data suggested that the complete loss ofYap1/Taz severely impairs hematopoietic development resulting in fetal death.

To continue investigating whether the deficiency of Yap1/Taz affects adult HSC function, we generated a hematopoietic, inducible (Mx1-CreTg/-) Yap1f/f;Tazf/f mouse model . Deficiency of

Yap1/Taz was induced by administration of polyinositide:polycytidine (polyI:C, Figure 3.1A and

Supplemental Figure 3.1B). No significant changes were observed in peripheral blood (PB), (white blood cell, neutrophil, platelet, reticulocyte counts and hemoglobin level) one week after induced deletion of Yap1/Taz (Supplemental Figure 3.1C). Yap1/Taz hematopoiesis displayed no difference in bone marrow (BM) cellularity (Supplemental Figure 3.1C), however Yap1/Taz BM contains a 2-fold increase in the frequency of colony-forming-units (CFU, 26±7.5 vs 50±5.6 CFU per 1.5x104 BM cells; p<0.001) (Figure 3.1B). Yap1/Taz HSC exhibit a loss of quiescence with a concomitant increase in the frequency of HSC in the G1 and S/G2/M phases of the cell cycle

Δ/Δ Δ/Δ (G0: 88.8 ± 3.0% vs 57.7 ± 10.3%, p<0.001; for Mx1Cre;Wt and Mx1Cre;Yap1 ;Taz HSC respectively) (Figure 3.1C). Additionally, Yap1/Taz HSC displayed reduced reconstitution abilities during serial competitive repopulation, (Supplemental 3.1D). Despite equal BM cellularity after secondary reconstitution, Yap1/Taz deficiency resulted in a 50% decrease of BM HSC

(11,742 ± 3,670 vs. 5,335 ± 3,954 HSC, p<0.05) (Supplemental Figure 3.1E). Consistent with increased cellular proliferation and cell cycle progression of BM HSC/P (Figures 3.1B-C),

Yap1/Taz HSC/P were more susceptible to exhaustion upon serial myeloablation resulting from

5-fluorouracil (5-FU).Yap1/Taz mice succumb to BM failure significantly earlier than their Wt counterparts (12.5 vs. 20.5 days, p<0.05) with significantly lower absolute neutrophil counts in PB

(Figure 3.1D and Supplemental Figure 3.1F). Taken together, the genetic deletion of Yap1 and its homologue Taz in HSC resulted in loss of HSC quiescence and increased HSC/P proliferation leading towards exhaustion.

Unbiased transcriptomic analysis of Yap1/Taz HSC failed to identify differential regulation genes classically associated with Yap1 and its cofactors (TEAD, p73, ERBB4, Runx,

-catenin/Tbx5 or Egr-1), including Cyr61, Ctgf, Ankrd1, Myc, Gli2, Vimentin, Axl, Bax and Birc5 marker genes (Supplemental Figures 3.1G-H). Instead, RNA sequencing analysis revealed significant differential clustering among genes involved with regulation of the actin cytoskeleton and small GTPases (Supplemental Figure 3.1G). Noteworthy, Gene Ontology pathway analysis

of Yap1/Taz HSC pinpointed differentially regulated genes involved in several pathways that coordinate small GTPase activity, responses to mechanical stimulus and cell cycle (Figure

3.1E). GO pathways such as these validate the in vivo phenotype of Yap1/Taz HSC and suggests that the loss of quiescence might be linked with small GTPase activity. Consistent with unchanged Yap1 transcriptional targets in Yap1/Taz HSC, we observed that Yap1 is exclusively polarized within the cytosol of Wt HSC (Figure 3.1F). Cytosolic location and function of murine Yap1 requires phosphorylation at serine 112 (Zhao et al., 2007) and indeed, Yap1 co- localized with its phosphorylated form (Figure 3.1F), indicating that cytosolic Yap1 is the predominantly featured in polarized BM HSC. In concordance with these functional and molecular data, deletion of Yap1 and Taz influence HSC fate at the division level. Analysis of molecular fate determinant allocation within paired daughter cells provides evidence that

Yap1/Taz HSC have an increased preponderance of commitment divisions and concordantly, decreased symmetric self-renewal divisions (Figure 3.1G-H).

HSC Scribble scaffolds cytosolic Yap1 with upstream inhibitory components of the Hippo

Signaling pathway

Accumulating evidence has shown that apical–basal polarity proteins from the Par complex (Par3/Par6/αPKC)and Scribble complex (Scribble/Dlg/Lgl) have been implicated in the regulation of the Hippo signaling pathway, thereby controllingYap1/Taz localization and function in epithelial cells (Baumgartner et al., 2010; Cordenonsi et al., 2011; Enomoto and Igaki, 2011;

Huang et al., 2013; Llado et al., 2015; Mohseni et al., 2014; Skouloudaki et al., 2009; Zhou et al.,

2017). Our previous data have identified that the deficiency of the Par polarity complex member, atypical Protein Kinase C (aPKC), is dispensable for HSC activity under basal, stressed or regenerative hematopoiesis (Nayak et al., 2019; Sengupta et al., 2011). Thus, we turned our

attention towards the basal polarity complex member, Scribble, and provide the first evidence that

Scribble is asymmetrically distributed and polarized in Wt HSC (Figures 3.2A, B). Both Yap1/Taz contain a Class I PDZ binding motif (-LTWL-COO-) at their extreme carboxyl-terminus through which have been predicted to bind with Scribble (Sundell et al., 2018). Using single-cell proximity ligation analysis (PLA), we demonstrated that Scribble interacts with Yap1 in ~80% of HSC

(Figures 3.2C, D and Supplemental Figure 3.2A).

To understand the role of the aforementioned Yap1/Scribble complex in HSC, we complemented our epistasis studies by the development of a novel Scribble floxed animal model

(Supplemental Figure 3.2B-D). Given that components of the upstream Hippo signaling pathway,

Lats1/2 and Mst1/2, have been shown to interact with Scribble in non-hematopoietic tissues

(Cordenonsi et al., 2011; Liu et al., 2017; Zhu et al., 2016) we confirmed that Scribble co-polarizes with the activated (phosphorylated) upstream inhibitory kinase Lats1 in ~60% of Wt HSC (Figure

3E and Supplemental Figures 3.2E-G). The deletion of Scribble in HSC disrupted the phospho-

Lats1/Yap1 complex, permitting Yap1 to translocate into the nucleus (Figures 3.2E, F). Yap1 mRNA expression, which can be dependent on its own transcriptional activity, is 4-fold upregulated in Scribble HSC (Figure 3.2G). Importantly, Scribble remains polarized inYap1/Taz HSC, suggesting that Scribble acts upstream of Yap1 polarization (Supplemental

Figures 3.2H, I), to control its cytosolic localization.

The PDZ domain of Scribble is necessary for Yap1 cytoplasmic polarization while the

LRR domain of Scribble is required for active Lats recruitment

We then hypothesized that Scribble acts as a molecular scaffold to facilitate upstream

Hippo signaling and the maintenance of Yap1 cytosolic localization and function. To test this hypothesis, we generated structure/function mutants of Scribble (Audebert et al., 2004) in bicistronic (EF1-IRES-RFP) lentiviral expressing vectors (Figure 3.3A). We transduced LSK (Lin-

sca-1+ c-kit+) BM cells with an empty lentiviral vector, vectors expressing the full-length Scribble protein or structure function mutants of Scribble. The overexpression of full-length Scribble in Wt

LSK BM cells maintains Scribble /Yap1 cytoplasmic polarization (Figures 3.3B, D), while

Scribble LSK BM cells display predominantly translocated nuclear Yap1 (Figures 3.3C, D). The expression of full-length Scribble in Scribble LSK BM cells restored Scribble/Yap1 co- polarization in the cytosol, effectively preventing Yap1 translocation to the nucleus (Figures 3.3C,

D). Forced expression of the PDZ containing mutant of Scribble within Scribble LSK cells reverted the nuclear accumulation of Yap1 back to the Wt-like cytoplasmic polarized state

(Figures 3.3C, D). The N-terminal LRR domain of Scribble successfully recruited pLats in the cytosol however, was unable to revert the nuclear Yap1 translocation in Scribble LSK BM cells

(Figures 3.3C-F). These data suggest that both Scribble PDZ domains and LRR domains are independently required for scaffolding Yap1 in proximity to its inhibitory kinase, Lats1. As expected, the expression of the extreme carboxyl-terminus section of Scribble (lacking any functional domains) cannot restore the co-polarization of Scribble with Yap1. Taken together,

Scribble is indispensable for Hippo mediated cytosolic Yap1 activity in HSC.

Scribble Scaffolds Polarized Yap1 and Activated Cdc42

To understand further the requirement of Yap1 in HSC we performed transcriptomic analysis on Wt and Scribble HSC. RNA-sequencing and GO pathway analysis revealed significant differential clustering among genes pertaining to protein binding, actomyosin formation and regulation of Rho GTPase activity, particularly Cdc42 (Supplemental Figures 3.3A-C). This observation parallels recent mass spectroscopy data on putative Scribble interacting partners as regulators of the cytoskeleton network and GTPase activity from hematopoietic cell lines (Mohr et al., 2018). To further funnel our analysis we identified 650 overlapping genes (~1/3 of all differentially expressed genes) when comparing the differentially regulated transcripts from

Scribble HSC with those of Yap1/Taz HSC (Figure 3.4A). Pathway analysis run on the overlapping gene set highlighted pathways regulating small GTPase activity (Figures 3.4B, C).

The GTPase and Rho guanyl-nucleotide exchange factor (GEF) activity genes include many upregulated GEFs that act specifically on RhoA, like Arhgef4, Arhgef28, Plekhg1 and Plekhg6

(Muller et al., 2018) along with other GEFs that favor Cdc42 like Fgd2, Fgd3, Fgd4, Mcf2l and

Arhgef4 (Figure 3.4C and Supplemental Figure 3.3C) (Muller et al., 2018). Noticeably, Rac1 specific GEFs (Prex1, Prex2, Plekhg6) (Muller et al., 2018) cluster together and display a modest upregulation in comparison to the upregulation of Cdc42 specific GEFs observed in theYap1/Taz HSC (Figure 3.4C). Given that both Scribble and Yap1 have independently been reported to regulate Cdc42 activation (Lim et al., 2017; Sakabe et al., 2017) with the aforementioned data, we hypothesized that the scaffolding ability of Scribble on Yap1 coordinates

Cdc42 activity and contributes to HSC function.

Active Cdc42 is a crucial regulator in HSC aging and specifically Cdc42 allocation accurately predicts asymmetric potential and fate (Florian et al., 2012; Florian et al., 2018; Florian et al., 2013; Schreck et al., 2017). We observed a significant (~60%) decrease in Cdc42-GTP

(active) levels after induced deletion of Scribble in BM progenitors through activated effector PAK pull down experiments (Supplemental Figures 3.3 D, E). To determine whether the changes in

Cdc42 activity corresponded with the scaffolding ability of Scribble over Yap1, we analyzed protein interactions between Scribble and Cdc42/Cdc42-GTP, as well as for Yap1 and

Cdc42/Cdc42-GTP through PLA. We report interactions of polarized Scribble with Cdc42 and

Cdc42-GTP (Gur-Cohen et al., 2015; Hu et al., 2017)) for 80% of HSC that were completely lost upon deficiency of Scribble (Supplemental Figures 3.3 G-I). The Yap1/Cdc42 and Yap1/Cdc42-

GTP complexes were present in the majority of HSC (~60% of HSC) in which both proteins complex in an asymmetric polarized manner (Figures 3.4 D-F). Upon loss of Scribble, the frequency of HSC which displayed a co-polarized state were significantly reduced by half, while

HSC acquired a non-polar interaction state or even no PLA signal, indicating a loss of proximity between Yap1 and Cdc42/Cdc42-GTP (Figures 3.4 D-F). Taken together, distinct cellular polarization states were identified in which Scribble is required for polarized cytoplasmic distribution of Yap1 with Cdc42 in HSC (Figure 3.4G).

HSC Scribble deficiency results in enhanced long-term self-renewal capacity

Despite the Scribble-dependent changes in the polarization of Yap1 with Cdc42 in HSC,

Hematopoietic-specific, constitutive Scribble- HSC did not display any significant changes in

PB white blood cell (WBC), neutrophil (ANE) and reticulocyte counts under basal hematopoietic activity (data not shown), similar to a previous report (Mohr et al., 2018). We next examined HSC fitness with a serial competitive repopulation assay (Figure 3.5A). Analysis of the output hematopoiesis revealed a decreased reconstitution potential from Scribble BM during early stages (weeks 4-12) in primary transplant recipients (Figure 3.5B). However, the long-term repopulation of Scribble HSC was unchanged in PB (Figure 3.1B) and BM (Supplemental

Figure 3.4A, B). Additionally, we observed a significant expansion of HSC/P populations in the

BM of these primary recipients (Supplemental Figure 3.4B). A competitive advantage of

Scribble HSC becomes apparent and statistically significant in subsequent tertiary recipients

(Figure 3.5C, D; Supplemental Figures 3.4C, D) which phenocopies the effect of conditional deletion of Lgl1 (Heidel et al., 2013). Chimera levels varied dramatically between Wt tertiary recipients where we observed a myeloid lineage bias consistent with aged hematopoiesis

(Beerman et al., 2010; Dykstra et al., 2011; Sudo et al., 2000). However, the BM content of

Scribble chimeric mice was 16-fold higher in lymphoid primed multipotent progenitors (LMPP, p<0.001) (Supplemental Figure 3.4D) with increased multi-lineage differentiation potential in the

PB, spleen and BM (Supplemental Figures 3.4E-G). Spleen weights of these animals were similar

(Supplemental Figure 3.4H) after tertiary reconstitution and we detected no evidence of myeloproliferation. Thus, serial competitive transplantation reveals that Scribble  HSC have

increased long-term repopulation ability with persistent self-renewal and maintained multi-lineage differentiation potential.

We recapitulated these in vivo findings using stroma-dependent cobblestone area forming cell (CAFC) frequency analysis (Supplemental Figure 3.4I). Scribble BM contains a reduced frequency (~60%) in the number of day-7 CAFC, which parallels the lag in early reconstitution after primary transplant, while the frequency of late (day 28-35) CAFC (corresponding to HSC) is

3-5 fold increased over Wt BM (Supplemental Figure 3.4J), indicating enhanced self-renewal potential. Taken together, these experiments provide evidence that polarized Scribble plays a functional role in hematopoiesis, regulating HSC self-renewal abilities and overall fitness upon serial BM transplantation. Importantly, expression of a transcriptionally competent, gain-of- function Yap1 mutant (Yap1 S112A) in HSC (Supplemental Figure 3.4K-L) did not alter either survival when challenged with serial myeloablative stress (Supplemental Figure 3.4M) or HSC competition repopulation abilities, complementary with a previous report (Jansson and Larsson,

2012) (Supplemental Figure 3.4N). These data indicate that the functional phenotypes associated with Scribble HSC is not driven by active Yap-dependent nuclear functions.

Scribble deficiency decreases survival of nascent non self-renewing HSC clones

Similar to the loss of quiescence observed when both Yap1/Taz are deleted in HSC

 (Figure 3.1C), Scribble HSC showed a decrease in the BM content of G0 HSC and a concomitant increase in the content of cycling HSC in the S/G2 and M phases (G0: 93±2.6% vs

88±4.3%, p<0.05) (Supplemental Figure 3.5A). This loss of quiescence became more apparent when we analyzed 5-Bromodeoxycytidine (BrdU) incorporation and retention in vivo (Wilson et al., 2008) (Figure 3.5E). Scribble mice displayed a 50% reduction in the content of BrdU- retaining (also called “dormant” (Wilson et al., 2008)) BM HSC and HSC/P (BM HSC: 1,398±489

vs 674±211 cells; BM LSK: 3,649±242 vs 2,097±515 cells, p<0.001) (Figure 3.5F and

Supplemental Figure 3.5B).

As a method to assess division kinetics at the single cell level, we sorted individual HSC into single wells and monitored their divisional history up to 40 hours in vitro. Consistent with our in vivo bulk HSC findings, roughly 20% of Scribble HSC have already undergone one cellular division (yielding two cells within the same well) within 24 hours, whereas such an event for Wt

HSC is nearly unobservable (Ema et al., 2000; Suda et al., 1984) (Figure 3.5G). By 40 hours of culture, both Wt and Scribble HSC have reached similar levels of primary divisions

(Supplemental Figure 3.5C). Clonal analysis of these paired daughter cells revealed altered division modalities (fate decisions) among Scribble HSC (Figures 3.5H, I and Supplemental

Figure 3.5D). Scribble HSC were enriched in self-renewing divisions due to a depletion of asymmetric differentiation and symmetric differentiation/committed clones (Figure 3.5J and

Supplemental Figure 3.5E). Such a depletion correlates with increased apoptosis of HSC upon induced, tamoxifen-driven in vitro deletion of Scribble in Rosa26-CreERTi2-ScribbleBM HSC

(Figure 3.5K). Notably, the PDZ domain introduced into Scribble stem and progenitor cells successfully restores apoptosis levels to that of the Wt HSC/P transduced with the control empty vector. (Figure 3.5L). However, reconstitution with the LRR containing mutant fails to revert the apoptosis levels seen in the Scribble  cells (Figure 3.5L) indicating that the C-terminus domains of Scribble are required for the maintenance of surviving asymmetric divisions. Analysis of molecular fate determinant allocation within paired daughter cells prior to the observed apoptosis provides evidence that ScribbleΔ/Δ HSC indeed have increased symmetric self-renewal divisions in vivo, at the expense of asymmetric divisions (Figure 3.5M-N). These results support the concept of in vivo selection of self-renewing HSC clones in hematopoietic-specific Scribble deficiency. Collectively, our data proves that Scribble controls asymmetric division potential and fate through the PDZ mediated scaffolding of cytosolic Yap1 with Cdc42-GTP.

Additional deficiency of Scribble restores HSC fitness ofYap1/TazΔ/Δ HSC and associates with Rac activation

Given that the loss of Yap1/Taz severely diminishes HSC quiescence and survival after

5-FU (Figure 3.1) and that we have correlated such phenotypes with a scribble-dependent cytosolic sequestration of Yap1 to modulate Cdc42-GTP activity, we wondered if the loss of all three proteins, Yap1, Taz and Scribble, would result in further decreased quiescence with increased sensitivity to 5-FU. To our surprise, the triple deficiency significantly restored the CFU

4 formation (50±2.3 vs 35±2.7 CFU/1.5x10^ ,p<0.05; Figure 3.6A) and the level of quiescence (G0:

57.7±10.34% vs 72.8±2.97%, p<0.05; Figure 3.6B) observed in the double  scenario to Wt levels, and as a result, restored survival following serial administration of 5-FU ( median survival of 12.5 vs. 24 days , p<0.05; Figure 3.6C). These data indicate that the additional deficiency of

Scribble inYap1/Taz mutant HSC restores quiescence and HSC fitness.

To determine whether Cdc42 expression and/or activation were modified by the triple deficiency of Scribble/Yap1/Taz, we performed single-cell analysis of the localization of Cdc42 and Cdc42-GTP. Immunofluorescence revealed that the combined deficiency of Yap1/Taz reduces the levels of Cdc42-GTP by ~25% in HSC without affecting the expression level of Cdc42

(Figures 3.6D-F). Our data confirmed that the triple deficiency of Yap1, Taz and Scribble resulted in further decrease (~60%) in active Cdc42 expression with a comparable decrease of the overall

Cdc42 protein (Figures 3.6D-F), indicating that the additional deficiency of Scribble results also in loss of Cdc42 protein rather than exclusively loss of its activation.

Since Cdc42 activity was not restored like the function of triple deficient HSC, we compared the transcriptome of HSC from Mx1-Cre;WT, Mx1Cre;Yap1Δ/Δ;TazΔ/Δ and

Mx1Cre;Scribble Δ/Δ;Yap1Δ/Δ;Taz Δ/Δ to determine whether specific transcriptional programs are modulated in triple deficient HSC leading to the overall neutralizing functional effect,

(Supplemental Figure 3.6A). By applying a filter on the same set of genes found differentially expressed in Mx1Cre;Scribble Δ/Δ (Supplemental Figures 3.3A-C) and Mx1Cre;Yap1Δ/Δ;Taz Δ/Δ

(Figures 3.4A-C), we identified that the Cdc42-specific GEFs, like Fgd4 were largely neutralized in Mx1Cre;Scribble Δ/Δ;Yap1Δ/Δ;Taz Δ/Δ HSC (Figure 3.6G), and there was an upregulation of the expression of GEFs with positive activity on Rac proteins, specifically Prex1 (Muller et al., 2018)

(Supplemental Figure 3.6B). Consistent with this upregulation in guanine nucleotide exchange factor specific for Rac1, we observed a ~2-fold upregulation of active Rac (Rac-GTP, Figure 3.6H) and ~75% inhibition of Cdc42 activation (Cdc42-GTP, Figure 3.6I) in

Mx1Cre;ScribbleΔ/Δ;Yap1Δ/Δ;Taz Δ/Δ stem and progenitor cells. Increased Rac1 levels have recently been reported to increase HSC repopulating capacity both in vitro and in vivo (Quarmyne et al., 2015) supporting the thesis that the increased activation of Rac in triple deficient HSC is involved in the restoration of quiescence and animal survival after serial administration of 5-FU, confirming the complex interplay of the Rho GTPase subfamily of proteins in the control of HSC activity.

Discussion

In this report, we have identified that the combined deficiency of Yap1/Taz results in loss of HSC fitness evidenced by decreased competitive repopulation ability in serial transplantations and survival upon multiple cycles of 5-fluorouracil chemotherapy (Carnevalli et al., 2014; Cheng et al., 2000; Essers et al., 2009). This effect may be independent of transcriptional regulation by

Yap1 since the expression of an active mutant of Yap1 does not alter the hematopoietic reconstitution. Rather, the role of Yap1/Taz in HSC seems to relate to their effect on polarity- based cell division and differentiation decisions controlled by the polarity master regulator Cdc42.

However, the fact that the canonical signaling pathway downstream of Cdc42, aPKC/Par3/Par6

does not impair hematopoiesis (Sengupta et al., 2011) suggests that a mechanism of Yap1 regulation via direct phosphorylation by PKCζ (Llado et al., 2015) is dispensable in HSC where an alternative pathway may control HSC fitness. Our data provide evidence for a functional complex containing Scribble, Yap1, p-Lats and Cdc42/Cdc42-GTP in dividing HSC that controls quiescence, fate and fitness (Figure 3.6J).

Scribble deficiency in HSC results in loss of function of HSC activity in primary recipients of competitive grafts but results in hematopoietic gain-of-function in tertiary recipient animals, phenocopying the effects of Lgl1 deficiency (Heidel et al., 2013). The positive regulatory role of

Scribble in primary recipients seems to depend on a Lgl1 independent pathway as the LRR domains of Scribble (responsible for binding Lgl1). In this report, we identified Scribble as a

Yap1/Lats1 polarizing scaffold molecule in quiescent HSC. Upon forced division, deletion of

Scribble disrupts the polarized cytosolic placement of Yap1 and induces apoptosis of asymmetrically dividing HSC (while sparing self-renewal) both of which are restored by expression of the PDZ domain-containing mutant of Scribble. Interestingly, the amino-terminus

LRR domain of Scribble is required for polarized localization of the upstream negative regulator of Yap1, phosphorylated (Thr1079) Lats1, however this domain could not rescue Yap1 polarization and function alone. We also identified that cytosolic Scribble andYap1/Taz are required for activation of Cdc42 and the combined loss of Scribble, Yap1/Taz results in compensatory Rac activation and HSC fitness restoration.

The roles of Cdc42 and Rac in HSC self-renewal are well established. Gene-targeted deletion of Cdc42 in mice leads to an increase in the HSC/P population in the BM due to enhanced cell cycle progression leading to HSC exhaustion and impaired long-term engraftment potential

(Yang et al., 2007). Rac GTPases play a crucial role in homing, migration, interaction with the microenvironment, and long-term engraftment potential of HSC/Ps (Cancelas et al., 2005; Ghiaur et al., 2008; Gu et al., 2003; Jansen et al., 2005). Gain-of-function of Rac has been recently associated with increased HSC repopulating capacity (Quarmyne et al., 2015). Similarly, the gain

of function of RhoA prevents asymmetric distribution of activated p38 MAPK of regenerating HSC in the context of transplantation (Hinge et al., 2017). Our data strongly indicates that the deficiency of Yap1/Taz and Scribble commonly result in changes in the transcriptome of GEFs for Cdc42,

Rac or RhoA. These changes are consistent with compensatory upregulation of the expression of Cdc42 GEFs and modest upregulation of Rac GEFs in bothYap1/Taz and Scribble HSC.

Overall, the triple deficiency of Yap1/Taz/Scribble results in upregulation of the Rac specific GEF

Prex1 and restored expression of the Cdc42-specific GEF Fgd4 to Wt levels. A GST-PAK pulldown for Rac confirmed that the triple deficiency of Yap/Taz/Scribble does result in increased

Rac activity and decreased Cdc42 activity. Activated Rac mediated HSC quiescence would explain the restoration of hematopoietic fitness of Yap1/Taz/Scribble deficient mice undergoing serial 5-fluorouracil administration (Supplemental Figure 3.6C).

Our data provide the first genetic demonstration that Hippo-regulated Yap1 controls HSC quiescence and fitness while the combined Yap1/Taz/Scribble complex controls Cdc42 activity and HSC fate through survival of asymmetrically dividing HSC.

Material and Methods

Animals. All mouse strains were maintained at an Association for Assessment and Accreditation of Laboratory Animal Care accredited, specific-pathogen–free animal facility at Cincinnati

Children’s Hospital Medical Research Foundation, Cincinnati, under an Institutional Animal Care and Use Committee approved protocol. All mice were between 6 and 12 wk of age at the time of experimentation. Mice genotypes were determined by PCR analysis. Sequences of genotyping primers are available upon request.

Exons 2-8 of the Wt Scribble locus were conditionally targeted to create Scribble flox/flox alleles.

LoxP sites were introduced into the introns 1 and 8 flanking Scribble exons 2 through 8 in mouse embryonic stem cells, using a selectable neo cassette flanked by frt sites. Targeted stem cells were injected into blastocysts of C57BL/6J mice to obtain chimeric floxed mice. After germline transmission, the mice were crossed to C57BL/6J mice expressing flp recombinase to remove the neo cassette. Progeny containing floxed Scribble alleles lacking the neo cassette

(Scribbleflox/flox) were further backcrossed to C57BL/6J mice. Construction of the targeting vector, generation and injection of targeted stem cells, and subsequent generation of chimeric mice were performed by inGenious Targeting Laboratory (New York, NY).

Scribbleflox/flox mice were crossed with Mx1-CreTg/- (Kuhn et al., 1995; Mikkola et al., 2003) and

Vav1CreTg/- (Wong et al., 2006) to generate hematopoietic specific genotypes. The Scribble flox/flox mice were also crossed with an inducible Rosa26Cre-ERTi2Tg/- background for inducible ubiquitous deletion. Cre-mediated recombination causes a frame shift and early stop of Scribble protein translation.

Tazflox/flox;Yap1flox/flox (Sakabe et al., 2017) were crossed with Vav-CreTg/- and Mx1-CreTg/- animals.

Yap1 (S112A)Tg/- (Chen et al., 2015) animals were crossed with Rosa26Cre-ERTi2Tg/- mice.

Littermate mice from the same breeding pair were used in all experiments.

Induction of Cre recombinase expression in inducible transgenic animals was achieved by either

6 injections of polyinositide/cytidine (polyI:C) at an intraperitoneal dose of 10 mg/kg every other day in Mx1-CreTg/- animals or by administration of 1mg tamoxifen (10mg/mL) intraperitoneal twice a day for 4 days for the Rosa26Cre-ERTi2Tg/- animals.

Fluorescence activated cell sorting (FACS) and immunophenotypic analysis of HSPC.

Bone marrow cells were stained using a mixture of biotin-conjugated monoclonal anti-mouse lineage antibodies against CD45R (B220, Clone RA3-6B2), Gr-1 (Ly6G, Clone RB6-8C5), CD4

(Clone RM4-5), CD8a (Ly-2, Clone 53–6.7), Mac-1 (CD11b, CloneM1/70), CD3ε (Clone 145–

2C11), and TER119 (Ly-76) (all from Becton Dickenson (BD), Franklin Lakes, NJ). In a subsequent labeling step, the cells were incubated with a combination of streptavidin- allophycocyanin (APC)-Cy7 (BD), phycoerythrin (PE) Cy7 anti-mouse Sca-1 (Ly6A/E, clone D7;

BD), APC anti-mouse CD117 (c-kit, Clone 2B8; BD), PerCP-cy5.5 anti-mouse CD16/32 (Clone

2.4G2;BD) FITC anti-mouse CD48 (Clone HM481; BD) or BV605 anti-mouse CD48 (Clone

HM481;BD), eFluor 450 anti-mouse CD34 (Clone RAM34; eBioscience), PE anti-mouse CD135

(FLT3, CloneA2F10.1) or PE anti-mouse CD150 (Clone mShad150; eBioscience, Thermo Fisher,

Santa Clara, CA) antibodies. Analysis of bone marrow nuclear cells harvested from all genotypes were immunophenotypically defined by differential expression of cell surface antigens: Lin- C-kit+

Sca-1+/- CD135+/- CD34+/- CD48+/- CD150+/- CD16/32+/-.

Cell cycle analysis. Cell cycle analysis of LSK CD48- CD150+ HSC harvested from the BM was performed by FACS analysis of immunophenotypically identified cells (see above for staining procedure) and linear-mode quantification of incorporation of Hoechst 33342 (2mg/mL) and

PyroninY (0.25 mg/mL). Verapamil (100 µM) was added during staining and analysis to prevent extrusion of Hoechst 33342.

Confocal immunofluorescence microscopy analysis. HSC sorted (using BD FACSAria II) from all geneotypes were seeded onto fibronectin (RetroNectin catalog T100B, TAKARA BIO

INC.) coated glass chamber slides in culture medium containing mouse stem cell factor (SCF)

(100 ng/mL) and thrombopoietin (TPO) (100 ng/mL). Cells were cultured overnight (10-12 hours) at 37 degree Celsius and then fixed in 4% paraformaldehyde for 20 minutes at 4°C, permeabilized with 0.1% Triton X-100 (catalog T9284, Sigma-Aldrich) for 10 minutes at room temperature, and blocked with 5% protease free bovine serum albumin (BSA) in phosphate buffered saline (PBS) for one hour. The slides were stained with primary antibodies in 5% BSA at 4°C overnight: Goat anti-Scribble (Santa Cruz, sc11048), Rabbit anti-phospho-Lats1 (Thr1079) (Cell Signaling

Technologies, 8654), Mouse anti-Yap1 (Santa Cruz, sc398182), Mouse anti-Cdc42-GTP (New

East Biosciences, 26905) or Rabbit anti-Cdc42 (Cell Signaling Technologies, 11A11). The cells were washed with PBS twice and then labeled with secondary antibodies (from Invitrogen):

Donkey anti–rabbit Alexa Fluor 488 (catalog A21206), donkey anti-mouse Alexa Flour 546

(A10036), donkey anti-goat Alexa Fluor 568 (A11057), donkey anti-goat Alexa Fluor 647 (A21447) or donkey anti-mouse Alexa Flour 647 (A31571) at 1:500 v/v concentration for 2 hours at room temperature. Cells were washed with PBS and then slides were mounted using Prolong Gold

Antifade mounting media (Thermo Fisher Scientific, P36935) containing DAPI. The stained cells were analyzed by a LSM 710 confocal microscope system (Carl Zeiss) equipped with an inverted microscope (Observer Z1, Zeiss) using a Plan Apochromat ×63 1.4 NA oil immersion lens.

Stained cells were also imaged with a Nikon Ti-E Inverted A1R Confocal Microscope with GaAsp

PMTs, Resonant Scanner, Piezo Z-Drive, Andor iXon 888 EMCCD Widefield Camera. Images were analyzed and processed using NIS-Elements and Adobe Photoshop v7.

Proximity Ligation Assay (PLA). HSC were sorted onto fibronectin coated glass chamber slides and cultured overnight at 37°C as previously indicated above under Confocal

Immunofluorescence Microscopy Analysis. Cells were subsequently fixed and permeabilized in preparation for PLA (Sigma, Duolink® In Situ Detection Reagents Red - DUO92008). After blocking with 5% BSA, primary antibodies were added: Goat anti-Scribble (Santa Cruz, sc11048),

Mouse anti-Yap1 (Santa Cruz, sc398182), Rabbit anti-Yap1/Taz (Cell Signaling Technologies,

D24E4), Rabbit anti-Cdc42 (Cell Signaling Technologies, 11A11), Mouse anti Cdc42-GTP (New

East Biosciences, 26905) and incubated at 37°C for 2 hours. The cells were washed and then treated with the PLA probe, a secondary antibody directly conjugated with oligonucleotides (PLA probe MINUS and PLA probe PLUS). A ligation solution, consisting of two oligonucleotides and

Ligase, is added and the oligonucleotides will hybridize to the PLA probe and join to form a circle if in close proximity. An amplification solution is added containing fluorescently labeled oligonucleotides and a polymerase. Here, the oligonucleotide arm of one PLA probe acts as a primer for a rolling-circle amplification (RCA) reaction using the ligated circles as a template, generating a repeated sequence product. The fluorescently labeled oligonucleotides then hybridize to the RCA product which is analyzed by fluorescent microscopy.

Paired Daughter Cell Assay. Isolate BMNC and preform Lineage cell depletion following the protocol for magnetic activated cell sorting (MACS) from Miltenyi Biotec (130-090-858). Stain lineage depleted HSPC as mentioned above for LSK CD48- CD150+ SLAM HSC. Sort single HSC, using BD FACSAria II, into individual wells of a Terasaki plate (Greiner Bio-One, 653108) with 17

µul of Stemspan (Stem Cell Technologies, 09650) medium containing SCF and TPO (100ng/ml).

After several hours of incubation at 37°C, wells with single cells were marked and monitored for cellular division at 24 and 40 hours post sort. At 40 hours, paired daughter cells were physically separated and deposited into individual wells of a 96 well round bottom tissue culture plate

(Falcon, 351177) containing 200 µL of Iscove's Modified Dulbecco's Medium (IMDM) (Thermo

Fisher Scientific, 12440053) supplemented with 10% fetal bovine serum (GE Healthcare

Lifesciences, Hyclone SH30396.03) plus SCF, TPO, G-CSF (20 ng/ml each), IL3 (50 ng/ml) and

EPO (4 U/ml) to allow four lineage myeloid differentiation. After 14 days of culture, paired clones were harvested for cytospin and stained with hematoxylin and eosin. Clones were examined for the presence of neutrophils (n), erythroid cells (e), macrophages (m) and megakaryocytes (M).

Colony-forming-cell assays. Hematopoietic stem and progenitor cells isolated from BM were grown on methylcellulose medium supplemented with cytokine mixtures (Methocult GF M3434;

Stem Cell Technologies) and colony-forming progenitors were scored on day 10.

Cobblestone Area Forming Cell (CAFC) assay. Murine BM cells are overlaid on flask bone marrow derived (FBMD-1) stromal cell layers in 12 dilutions, 2-fold apart (15 wells/dilution), to allow limiting dilution analysis of the precursor cells forming hematopoietic clones. Cultures are fed weekly by changing half of the medium MyeloCult M5300 base (Stem Cell Technologies,

05350) supplemented with 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 10 µM hydrocortisone (hydrocortisone-21-hemisuccinate, Sigma-H4001)] while frequencies of CAFC are assessed at weekly intervals. Wells were scored positive if at least one phase-dark hematopoietic clone (containing 5 or more cells) is observed. The frequency of CAFC was calculated using limiting dilution analysis software (L-Calc, version 1.1) and Poisson statistics.

Longterm BrdU incorporation and retention assay (Wilson et al., 2008). Animals freely imbibed water containing 5-bromodeoxycytidine (BrdU: 1mg/mL) and 5% glucose for 10 days.

Animals were euthanized after 10 days of BrdU administration to assess levels of incorporation into HSPC populations as well as 80 days post BrdU administration to quantify quiescence within

HSPC populations determined by BrdU retention (BD Pharmingen intracellular staining kit: Anti-

BrdU-Alexa 488).

Serial competitive repopulation transplantation. Equal amounts of BM from CD45.2+ wild-type

(Wt) and ScribbleΔ/Δ mice were mixed with congenic CD45.1+ B6.SJLPtprcaPep3b/BoyJ competitor cells and were transplanted at a 1:1 ratio into lethally irradiated B6.SJLPtprcaPep3b/BoyJ recipients.

Peripheral blood chimera (measured by CD45.2+ leukocytes) of primary, secondary and tertiary recipients were measured every 4 weeks and absolute blood counts were recorded. Peripheral blood FACS analysis of CD45.2 chimera and lineage reconstitution was performed by staining peripheral blood cells using monoclonal anti-mouse lineage antibodies against CD45.2 (Clone

104), CD45.1 (Clone A20), CD45R (B220, Clone RA3-6B2), CD11b (M1/70) and CD3ε (Clone

145-2C11). After 16 weeks of reconstitution, recipients were sacrificed and peripheral blood, BM and spleen were analyzed for total CD45.2 chimera, lineage reconstitution/ absolute lineage positive cellularity and HSPC analysis was performed on BM and spleen. BM cells were pooled from each recipient for each genotype and serially transplanted into secondary recipients. This process was terminated after 16-20 weeks of tertiary reconstitution.

5- Fluorouracil (5-FU) administration and survival analyses. Kaplan-Meier survival analysis of mice after myeloablation with serial injections of 5-fluorouracil (150 mg/kg) five days apart.

Absolute neutrophil, platelet and reticulocyte counts were recorded every 5 days from retro-orbital blood draws. Histology of femurs and spleens were assessed at the time of death or sacrifice.

Generation of Scribble structure/function mutants and lentiviral transduction. Scribble structure and function mutants were generously provided by Jean-Paul Borg of Centre de

Recherche en Cancérologie de Marseille, in a pET-431b(+) vector fused at the amino terminus with the 491aa Nus•Tag™ protein (Audebert et al., 2004). We used polymerase chain reaction

(PCR) amplification to exclude the Nus tag from our Scribble sequences and subcloned into a stem cell specific EF1α-MCS-IRES-RFP lentiviral vector (System Bioscience, CD531A-2). The full-length Scribble sequence would not PCR amplify from the pET-431b expression vector with high purity and therefore, we digested the full-length cDNA from another Scribble containing plasmid (using EcoR I and Not I), and pasted it into our EF1α-MCS-IRES-RFP lentiviral vector.

All plasmids were verified by sequencing and confirmed to be in frame and have correct start and stop codons corresponding to the original constructs and cDNA. Sequences of cloning and genotyping primers are available upon request. We made virus with these plasmids with high titers and transduced hematopoietic stem and progenitor cells (HSP/C) with an MOI of 20.

Rac1/Cdc42 glutathione-S-transferase (GST) p21 activated kinase (PAK) effector pull down assay. Active Rac1/Cdc42 pulldowns were performed using the Rac1/Cdc42 Activation Magnetic

Beads Pulldown Assay Kit (Millipore, 2718273) on lineage depleted BM cells.

Transcriptome and bioinformatics analysis. Total RNA was extracted from sorted HSC derived from Mx1Cre;Wt or ScribbleΔ/Δ, TazΔ/Δ;Yap1Δ/Δ and ScribbleΔ/Δ;Taz Δ/Δ;Yap1Δ/Δ mice one week following polyinositide/cytidine (polyI:C) administration and gene deletion using RNeasy Mini Kit

(QIAGEN). RNA quality and concentration were measured by Bioanalyzer 2100 using the RNA

6000 Nano Assay. The initial amplification step was performed with the Ovation RNA-Seq System v2 (Tecan Genomics) and the cDNA concentration and size were measured using the Qubit dsDNA BR assay and a DNA 1000 Chip, respectively. The subsequent libraries were prepared with the Nextera XT DNA Sample Preparation Kit (Illumina Technologies). Briefly, 1ng of cDNA was suspended in Tagment DNA Buffer, and tagmentation (fragmentation and tagging with the

adaptors) was performed with the Nextera enzyme (Amplicon Tagment Mix), incubating at 55C for 10 min. NT Buffer was then added to neutralize the samples. Libraries were prepared by PCR with the Nextera PCR Master Mix, and 2 Nextera Indexes (N7XX, and N5XX) according to the following program: one cycle of 72°C for 3 min, one cycle of 98°C for 30 sec, 12 cycles of 95°C for 10 sec, 55°C for 30 sec, and 72°C for 1 min, and one cycle of 72°C for 5 min. The purified cDNA was captured on an Illumina flow cell for cluster generation and libraries were sequenced on the Illumina HiSeq2500 following the manufacturer's protocol. The concentration of the pool was optimized to acquire at least 25-30 million reads per sample using Paired-End Reads with a read length of 75 bps. Reads were aligned with TopHat software, using hg19 as the reference genome and reads per kilobase of transcript per million mapped reads (RPKM) as output. RPKM were log2-transformed and baselined to the median expression of the average of each class of samples. Analysis of differential gene expression and gene ontology (GO) pathway analyses using Altanalyze software (developed by Cincinnati Children’s Research Foundation).

Statistical analysis. Data are presented as average ± standard deviation. Comparisons were performed with Student t test, Chi-squared test and one-way or two-way ANOVA when required.

Statistical significance levels were established at 5%, 1% and 0.1%.

Acknowledgements

This study was supported (in part) by the founding from the National Institutes of Health

(F31HL132468) and the University of Cincinnati (Albert J Ryan Foundation and Cardell Fellowship for Excellence in Graduate Research), awarded to MJA.

Author contributions

MJA, RCN, SH, AMW, BB performed experiments. MJA and JAC analyzed data and wrote the manuscript. MJA and JAC designed experiments. MDF, MX, QRL, HG, YZ, MTD and JM provided critical reagents, insightful views and critiques and edited the manuscript. JAC led the group and supervised MJA.

Disclosure of conflicts of interest

The authors declare no relevant conflicts of interest

Figures and legends

Figure 3.1. Yap1/Taz are necessary for HSC function. (A) Schematic representing the inducible deletion of Yap1/Taz in the hematopoietic system followed by one week of recovery before subjecting mice to experimental testing. (B) Number of Colony Forming Units (CFU) from BM cells. (C) Cell cycle analysis of Lin- CD48- CD150+ HSC by FACS. (D) Kaplan-Meier survival analysis after serial myeloablation with 5-fluorouracil (150 mg/kg) five days apart. (E) Gene

Ontology (GO) pathway analysis of differentially regulated genes (p<0.05) between Mx1Cre;Wt and Mx1Cre;Yap1 Δ/Δ;TazΔ/Δ HSC, (cutoff: 1.5 fold). Numbers represent the percentage of genes within each GO pathway that are differentially regulated. (F) Immunofluorescence depicting Yap1 protein localization in Wt HSC (immunophenotypicaly defined as LSK CD150+ CD48-) (red pseudocolor) and Yap1 phosphorylation status at Serine 112. Cells are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (G)

Immunofluorescence depicting fate determinant allocation of Myc and the corresponding HSC division mode in Wt and Yap1/TazΔ/Δ paired daughter HSC, (Nocodazol 10nM for 24hours). Low

Myc expression in paired daughter cells represents symmetric self-renewal (i), high and low Myc expression between the two daughters represents an asymmetric division (ii), while high Myc expression in both cells is indicative of symmetric commitment (iii). (H) Quantification of fate determinant allocation and division mode among Wt and Yap1/TazΔ/Δ paired daughter HSC. *** p<0.05; *** p<0.001.

Figure 3.2. Scribble scaffolds components of the Hippo pathway in HSC and controls Yap1

cytoplasmic localization. (A) Immunofluorescence depicting Scribble protein localization in Wt

HSC (immunophenotypicaly defined as LSK CD150+ CD48-) (white areas). Cells are counterstained with DAPI and merged images are shown in the bottom micrographs. Scale bar is

5 µm. (B) Quantification for the frequency of HSC with Scribble polarization. (C)

Immunofluorescence depicting a Proximity Ligation Assay (PLA) on Wt and Scribble Δ/Δ HSC using anti-Scribble and anti-Yap1 primary antibodies subsequently targeted with corresponding probes for oligomerization. The detected dimers are pseudocolored in red. Nuclei are counterstained with

DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (D)

Quantification of PLA signal. (E) Immunofluorescence showing Scribble polarization and Yap1 co-localization (white arrow heads) in HSC isolated from Wt mice. White asterisks indicate areas of co-localization between Scribble and the activated upstream inhibitory kinase of Yap1, phosphorylated Lats1/2. White arrows denote Yap1 nuclear translocation in Scribble Δ/Δ HSC.

Nuclei are counterstained with DAPI and merged images are shown in the right micrographs.

Scale bar is 5 µm. (F) Quantification for the frequency of HSC with Yap1 nuclear foci. (G)

Quantitative RT-PCR of Yap1 mRNA expression from HSC cultured for 40 hours, ** p<0.05;

**p<0.01.

Figure 3.3. Cytoplasmic polarization of Yap1 is restored in ScribbleΔ/Δ HSC/P with expression of Full length Scribble or PDZ containing mutants. (A) Graphical representation of the functional domains in human full length scribble protein and the truncation mutations incorporated into an Ef1α-IRES-RFP lentivirous. (B) Immunofluorescence showing Scribble polarization and Yap1 co-localization in Lin- Sca-1+ c-kit+ (LSK) BM cells isolated from Wt mice and transduced with EMPTY (Ef1α-IRES-RFP) lentivirus or human full length Scribble as indicated. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (C) Immunofluorescence showing Scribble expression and Yap1

Δ/Δ localization in Lin- Sca-1+ c-kit+ BM cells isolated from Scribble mice and transduced with

EMPTY (Ef1α-IRES-RFP) lentivirus, human full length Scribble or structure-function mutants as indicated. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (D) Quantifications for the frequency of transduced LSK cells with nuclear Yap1. (E) Immunofluorescence showing Scribble and pLats1 expression in Lin- Sca-1+ c-

Δ/Δ kit+ BM cells isolated from Scribble mice and transduced with EMPTY (Ef1α-IRES-RFP) lentivirus or the LRR mutant. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (F) Quantification of pLats1 colocalization with

Scribble when the N-terminal portion of Scribble (LRR) is reintroduced into Scribble null LSK cells.

(G) Cartoon depicting the ternary complex between Scribble, activated Last1/2 and Yap1 in the cytosol of Wt HSC. ** p<0.01; *** p<0.001.

Figure 3.4. Scribble scaffolds Yap1 and Cdc42 in the cytoplasm of HSC. (A) Venn Diagram highlighting the number of differentially regulated genes in common when comparing Mx1Cre;Wt and Mx1Cre;Scribble Δ/Δ and comparing Mx1Cre;WT and Mx1Cre;Yap1Δ/Δ;TazΔ/Δ HSC. (B) Gene

Ontology (GO) pathway analysis of the common differentially regulated genes between two independent analysis of Mx1Cre;Wt to Mx1Cre;ScribbleΔ/Δ HSC and of Mx1Cre;Wt to

Mx1Cre;Yap1Δ/Δ;TazΔ/Δ HSC . Numbers represent the number of genes within each GO pathway that are differentially regulated. (C) Heat map depicting the differential regulation of common genes between Mx1Cre; ScribbleΔ/Δ and Mx1Cre;Yap1Δ/Δ;TazΔ/Δ HSC that pertain to the Rho guanyl nucleotide exchange factor activity and small GTPase activity gene ontology pathways.

(D-E) Proximity Ligation Assay (PLA) detection of endogenous Yap1/Cdc42 (D) and Yap1/Cdc42-

GTP (E) interactions in HSC. The detected dimers are represented by fluorescent dots (red).

Nuclei are counterstained with DAPI and merged images are shown in the right micrographs.

Scale bar is 5 µm. (F) Frequency of HSC in which PLA signal depicted in D and E was found in relation or not with polarization. *p<0.05 and ** p<0.01 between Vav1Cre;WT and

Vav1Cre;ScribbleΔ/Δ; ## p<0.05 between Cdc42 and Cdc42-GTP frequencies. (G) Cartoon representing the overall polarization status between Scribble, Yap1 and Cdc42 in Wt cells emphasizing the loss of co-polarization and asymmetry in Scribble  HSC.

Figure 3.5. ScribbleΔ/Δ hematopoietic reconstitution develops a competitive advantage when serially transplanted by maintaining self-renewal divisions. (A) Schematic representing a serial competitive repopulation assay (CRA). Equal amounts of BM from CD45.2+

Vav1Cre Wt and Scribble null mice mixed with congenic CD45.1+ B6.SJLPtprcaPep3b/BoyJ competitor cells were transplanted at a 1:1 ratio into lethally irradiated B6.SJLPtprcaPep3b/BoyJ recipients. (B-D)

PB chimera (CD45.2+ leukocytes) of primary (B), secondary (C) and tertiary (D) recipients. (E)

Schematic representing a long-term BrdU incorporation assay in which animals freely imbibed water containing 5-Bromodeoxycytidine (BrdU: 1mg/mL) for 10 days. Animals were euthanized after 80 days post BrdU administration to quantify quiescence within hematopoietic stem and progenitor (HSC/P) populations was determined by BrdU retention (BD Pharmingen intracellular staining kit: Anti-Brdu, Alexa 488). (F) Absolute BrdU retaining (BrdU+) HSC assessed by FACS analysis of BM from mice as described in E. (G) Division of sorted, and individually deposited

HSC depicting the averages and standard deviations of the relative number (%) of wells containing the indicated number of cells after 24 hours in culture. (Four independent experiments, n>450 HSC). (H) Schematic of an in vitro Paired Daughter Cell Assay to assess fate decisions among individually sorted HSC. (I) Representative cytospin images of paired daughters, m=macrophage, n=neutrophil, e=erythrocyte and M=megakaryocyte. (J) Absolute number of paired daughter cells analyzed for division modality, assessed as the presence or absence of full multi-lineage differentiation potential among individual paired daughter clones. n of paired daughter separations >200. Chi Square analysis. (K) Cell death analysis using Annexin V and 7-

AAD staining on Rosa26Cre;Scribblefl/fl cells after 40 hours of culture with 4-OH Tamoxifen. (L)

Cell death analysis using Annexin V staining in Lin- Sca-1+ c-kit+ BM cells isolated from

Δ/Δ Vav1Cre;Wt or Vav1Cre;Scribble mice transduced with EMPTY (Ef1α-IRES-RFP) lentivirus or

Scribble structure-function mutants as indicated. (M) Immunofluorescence depicting fate determinant allocation of Myc and the corresponding HSC division mode in ScribbleΔ/Δ paired

daughter HSC, (Nocodazol 10nM for 24hours). Low Myc expression in paired daughter cells represents symmetric self-renewal (i), high and low Myc expression between the two daughters represents an asymmetric division (ii), while high Myc expression in both cells is indicative of symmetric commitment (iii). (N) Quantification of fate determinant allocation and division mode among ScribbleΔ/Δ paired daughter HSC. *p<0.05, **p<0.01 and ***p<0.001.

Figure 3.6. Deficiency of Scribble restores HSC fitness of Yap1/TazΔ/Δ HSC and associates with Rac activation. (A) Number of Colony Forming Units (CFU) from BM cells, *** p<0.001, * p<0.05. (B) Cell cycle analysis of Lin- CD48- CD150+ HSC harvested from the BM of Mx1Cre Wt,

Δ/Δ TazΔ/Δ;Yap1Δ/Δ and Scribble ;TazΔ/Δ;Yap1Δ/Δ mice one week after Poly I:C. Stages of cell cycle were assessed by FACS analysis with incorporation of DNA binding Hoechst 33342 (2mg/mL) and nucleotide binding PyroninY (0.25mg/mL). (C) Kaplan-Meier survival analysis after serial myeloablation with 5-fluorouracil (150mg/kg) five days apart.(D) Immunofluorescence of HSC showing Cdc42 expression and localization along with Active Cdc42-GTP expression and localization. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar 5 µm. (E) Quantification of Cdc42 expression measured by MFI. (F)

Quantification of Cdc42-GTP expression measured by MFI. (G) Heat map depicting genes within the Rho guanyl nucleotide exchange factor activity and small GTPase activity gene ontology pathways showing differential regulation between Mx1Cre Wt, TazΔ/Δ;Yap1Δ/Δ and

Δ/Δ Scribble ;TazΔ/Δ;Yap1Δ/Δ HSC. (H) Rac1/Cdc42 activation PAK Pulldown on Lineage depleted

- (Lin-) BM cells. (I) Cdc42 effector PAK pull down from of Wt and Scribble Δ/Δ Lin BM cells. (J)

Cartoon depicting a complete picture of the Scribble polarity complex and its components in relation with HSC self-renewal and cell cycle entry. *p<0.05, **p<0.01 and ***p<0.001.

Supplementary Figures and Tables

Supplemental Figure 3.1. Constitutively transcriptionally active Yap1 function is dispensable for HSC functional activity. (A) Non-Mendelian inheritance of Vav1CreTg/-

;TazΔ/Δ;Yap1Δ/Δ mice. Predicted Vav1CreTg/-;TazΔ/Δ;Yap1Δ/Δ mice are not viable. (B) Genomic PCR showing inducible (Mx1Cre) deletion of Yap1 and Taz in bone marrow. (C) Hematopoietic cellularity within the peripheral blood (PB) and bone marrow of Mx1Cre;Wt and

Mx1Cre;TazΔ/Δ;Yap1Δ/Δ mice one week after induced deletion. PB counts were measured by white blood cell (WBC), absolute neutrophil (ANC), platelet and reticulocyte counts. (D) Serial

Competitive Repopulation Assay between Wt and TazΔ/Δ;Yap1Δ/Δ deficient hematopoietic reconstitution. Deletion was induced by Poly I:C injections (10mg/kg) 4 weeks after transplant and subsequent data time points are normalized to the pre-Poly I:C chimera at week 4. (E) Absolute number of BM CD45.2+ cellularity 16 weeks after secondary reconstitution. (F) PB ANC of

Mx1Cre;Wt and Mx1Cre;TazΔ/Δ;Yap1Δ/Δ mice at different points after serial 5-FU administration.

(G) RNA-sequencing heat map clustering significant differential regulation between Mx1Cre;Wt and Mx1Cre;Yap1 Δ/Δ;TazΔ/Δ HSC. ***p<0.001. (H) Normalized expression values (mRNA) for pre- specified Yap driven transcripts between Mx1Cre;Wt and Mx1Cre;Yap1Δ/Δ;TazΔ/Δ HSC from

RNAseq analysis. n.d.= not detected.

Supplemental Figure 3.2. Scribble deficient HSC lose polarized Yap1 expression in the cytosol. (A) Immunofluorescence depicting Proximity Ligation Assay on Wt HSC using IgG control primary antibodies (anti-goat IgG and anti-Mouse IgG) subsequently targeted with corresponding probes for oligomerization. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (B) Generation of Scribble Δ/Δ mice:

Exons 2-8 of the Wt Scribble locus were conditionally targeted to create Scribbleflox/flox alleles and crossed with Mx1-Cre Tg/- and Vav1Cre Tg/- to generate hematopoietic specific genotypes. (C) qRT-

PCR of Scribble mRNA from bone marrow (BM) HSC (immunphenotypically defined and sorted

- + + - + by differential expression of cell surface antigens: Lin c-kit Sca-1 CD48 CD150 ) isolated from

Δ/Δ Vav1CreTg/- Wt or Scribble animals. *** p<0.001. (D) Western blot analysis of Scribble protein in

Δ/Δ bone marrow nucleated cells (BMNC) isolated from Vav1Cre Tg/- Wt and Scribble mice. Top arrow denotes the full-length protein and the bottom two arrows are likely expression of translated splice variants. (E-G) Quantification of HSC Immunofluorescence depicting frequency of HSC with polarized Scribble (F) Scribble-Yap1 co-localization (G) and Scribble-pLats co-localization.

(H) Immunofluorescence showing Scribble localization in Wt and TazΔ/Δ;Yap1Δ/Δ HSC. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5

µm. (I) Quantification of the frequency of HSC with Scribble expression and polarization. *p<0.05.

Supplemental Figure 3.3 Scribble binds to and modulates Cdc42 expression and activation. (A) RNA-sequencing heat map clustering significant differential regulation between

Mx1Cre;Wt and Mx1Cre;ScribbleΔ/Δ HSC. (B) Gene Ontology analysis of differentially regulated pathways between Mx1Cre;Wt and Mx1Cre;ScribbleΔ/ΔHSC, 1.5 fold. Numbers represent the number of genes within each GO pathway that are differentially regulated. (C) Heatmap clustering statistically significant genes from small GTPase binding and Rho guanyl-nucleotide exchange

- factor activity signatures. (D) Cdc42 effector PAK pull down from of Wt and Scribble deficient Lin

BM cells. (E) Quantification of total and active Cdc42 expression from Lin- BM cells measuring band intensity preformed using Image J software comparing Cdc42 or Cdc42-GTP to β-actin levels. (F-G) PLA detection of endogenous Scribble/Cdc42 (F) and Scribble/Cdc42-GTP (G) interactions in HSC. The specificity of the interactions is revealed by the reduced signal detected in Scribble deficient HSC. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. Scale bar is 5µm. (H) Quantification of PLA signal from

F and G. *** p<0.001.

Supplemental Figure 3.4 Scribble deficiency increases repopulation and self-renewal of

HSC. (A) Bone Marrow (BM) chimerism measured by CD54.2+ BM nuclear cells (BMNC) from primary recipients of Vav1Cre serial transplant. (B) BM CD45.2+ LSK cellularity from primary recipients. *p<0.05. (C) BM chimerism from tertiary recipients of Vav1Cre serial transplant.

**p<0.01. (D) BM CD45.2+ LSK cellularity from tertiary recipients. *p<0.05. (E-G) Lineage reconstitution among tertiary recipients from the PB (E), BM (F) and Spleen (G). *p<0.05,

***p<0.001. (H) Spleen weight (grams) from the tertiary recipient mice. (I) Schematic depicting a cobblestone area forming cell (CAFC) assay where murine BM cells are overlaid on a Flask Bone

Marrow Derived (FBMD-1) stromal cell monolayer in 12 dilutions, 2-fold apart, to allow limiting dilution analysis of the precursor cells forming hematopoietic clones. (J) Cobblestone area forming ability after several weeks in culture. The frequency of CAFC was calculated using

Poisson statistics. ***p<0.001. (K) Immunofluorescence showing overexpression and nuclear accumulation of Yap1 (S112A) in HSC. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar is 5 µm. (L) Quantification for the frequency of HSC with nuclear Yap1 expression. (M) Kaplan-Meier survival analysis of Wt and Yap1 (S112A) transgenic mice after serial myeloablation with 5-fluorouracil (150mg/kg). (N) Serial Competitive

Repopulation Assay between Wt and Yap1 (S112A) overexpressing hematopoietic reconstitution.

Supplemental Figure 3.5 Scribble deficiency decreases quiescence of HSC and modulates fate. (A) Cell cycle analysis of Lin- CD48- CD150+ HSC harvested from the BM of Wt and

Δ/Δ Scribble mice. Stages of cell cycle were assessed by FACS analysis with incorporation of DNA binding Hoechst 33342 (2 mg/mL) and nucleotide binding PyroninY (0.25mg/mL). (B) Absolute

BrdU retaining (BrdU+) LSK cells assessed by FACS analysis of BM from mice described in

Figure 5E. ***p<0.001. (C) Division of sorted, and individually deposited HSC. Figure depicts the averages and standard deviations of the relative number (%) of wells containing the indicated number of cells after 40 hours in culture. (Four independent experiments, n>450 HSC). (D)

Representative cytospin images of HSC fate or division modality, m=macrophage, n=neutrophil, e=erythrocyte and M=megakaryocyte. Symmetric Self-Renewal division is indicated by the presence of all 4 lineages (macrophage, neutrophil, erythrocyte and megakaryocyte) in each clone/ daughter; Asymmetric Division occurs when one clone is missing one or more of the 4 lineages; while symmetric Differentiation / Commitment is determined when both clones are incomplete and missing one or more lineage output populations. (E) Relative frequency of paired daughter cells analyzed for division modality, assessed as the presence or absence of full multi- lineage differentiation potential among individual paired daughter clones. n of paired daughter separations ~100. Chi Square analysis; ** p<0.01.

Δ/Δ between Mx1Cre Wt, TazΔ/Δ;Yap1Δ/Δ and Scribble ;TazΔ/Δ;Yap1Δ/Δ HSC. (B) Expression (mRNA)

Δ/Δ Δ/Δ of Prex1 and Fgd4 in Mx1Cre Wt, Scribble , TazΔ/Δ;Yap1Δ/Δ and Scribble ;TazΔ/Δ;Yap1Δ/Δ HSC.

* p<0.05; ** p<0.01; *** p<0.001. (C) Cartoon depicting the major functional consequences on

HSC activity upon loss of Yap/Taz (top panel), Scribble (middle panel) and Scribble/Yap/Taz

(bottom panel).

Chapter 4: Scribble mediates IFN-I induced activation of HSC through its regulation of Sca-1 and Akt activity independent of Stat1 effector response.

Mark J. Althoff1,2,3, Ashley M. Wellendorf1, Maria T. Diaz-Meco4, Jorge Moscat4, and Jose A. Cancelas1,2,3

Manuscript is in preparation.

Abstract

Hematopoietic stem cells (HSC) are highly quiescent cells with the ability to rapidly enter the cell cycle and differentiate through changes in their polarity and the disposition of intracellular molecular fate determinants in response to microenvironment (ME) cues. IFN-I are microenvironment cytokines produced during the physiological response mounted to combat a viral infection. In bone marrow hematopoiesis, IFN-I induce proliferation of HSC. Clinically, patients treated with IFN-I, as well as individuals suffering from IFN-I associated chronic disease, often exhibit sustained hematological cytopenias and HSC failure. The precise molecular mechanisms that govern HSC behavior in response to IFN-I are still unclear. Our data highlights that the deficiency of Scribble in HSC rendered them less sensitive to IFN-I mediated activation.

As a result, Scribble deficient HSC exposed to acute IFN-I stimulation are functionally more fit, displaying increased competitive reconstitution abilities during serial transplantations. No discernible differences in Stat-1 (a major effector of IFN-I signaling) activity were observed when measuring phosphorylation status, nuclear translocation and transcriptional response within Wt and Scribble deficient HSC following IFN-I exposure. Ly6a transcript levels are appropriately upregulated following IFN-I stimulation, however the encoded stem cell antigen-1 (Sca-1) protein localization was significantly decreased on the membrane surface. We are keen on further understanding the role of Scribble in coordinating HSC endosomal membrane trafficking and how it relates to IFN-I mediated HSC activation

Introduction

The potential of hematopoietic stem cells (HSC) to reconstitute the hematopoietic system has allowed for the development of transplantation approaches to treat cancer and genetic diseases. Cell cycle status of HSC defines their ability to engraft in conditioned recipients. Only quiescent HSC (qHSC) retain the necessary reconstitution and long-term self-renewal abilities required for transplantation (Fleming et al., 1993). qHSC can rapidly respond to cues produced by the environment following hematopoietic stress or damage, and exit from quiescence to replenish blood cells. Therefore, one of the most striking features of HSC is their ability to adapt to the changing needs of the blood system. Each of the different cellular states in HSC (i.e., quiescence, proliferation, and differentiation) imposes a unique set of transcriptional demands

(Shyh-Chang et al., 2013). The cellular states of HSC depend on instructional cues from the surrounding marrow microenvironment (ME) that activate or inhibit their proliferation (Schuettpelz and Link, 2013).

Interferons type I (IFN-I) are ME cytokines produced by a variety of immune and nonimmune cells that mediate host immune responses to viruses and intracellular pathogens

(Platanias, 2005). The recognized positive values of IFN-I allow their use for therapy of viral infections, myeloproliferative disorders, myeloid malignancies such as chronic myelogenous leukemia (CML) and autoimmune diseases. However, high levels of exogenous or endogenous

IFN-I result in HSC failure through only partly known mechanisms (Gokce et al., 2012). In bone marrow (BM) hematopoiesis, IFN-I have been shown to be a crucial positive regulator for proliferation of qHSC (Essers et al., 2009). These findings provide novel insight for understanding the success IFN-I has in treating chemotherapy-resistant HSC-derived leukemias like CML. CML patients treated with IFN-I prior to imatinib (molecularly targeted therapy directed against BCR-

ABL, the fusion protein characteristic of CML) experienced sustained remission upon drug discontinuation (Krause and Van Etten, 2008; Rousselot et al., 2007) . Similar remission was

established among patients from the same study who received imatinib without prior IFN-I treatment however, these patients often relapsed upon imatinib withdrawal. Here, IFN-I are believed to have induced proliferation of a persistent CML initiating/ quiescent leukemic stem cell population rendering them vulnerable to targeted therapy with imatinib. Such findings in the field are monumental and provide enticing new treatment strategies for CML and other drug-resistant hematological malignancies. However, we lack knowledge of the fundamental molecular mechanisms that orchestrate this phenomenon. Understanding of the molecular mechanisms that transduce IFN-I signals in qHSC will define a therapeutic window and identify biomarkers of activity.

Cellular polarity is believed to be required for HSC to maintain quiescence as well as control the precise balance of self-renewal and multi-lineage differentiation, ensuring appropriate numbers of both stem cells and committed differentiated cells during tissue development, homeostasis and repair. Although cellular polarity is a vital factor regulating HSC activity, the role polarity regulators have on HSC polarization and function is largely understudied. The Scribble

Complex, consisting of Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble, is one of three major polarity complexes conserved throughout Metazoan phylogeny and controls the spatial organization of intracellular proteins. Human Scribble was identified as a specific target, via PDZ protein interactions, for the HPV E6 oncoprotein (Nakagawa and Huibregtse, 2000). The targeted depletion of Scribble in a subset of cervical cancers supports the possible function of Scribble in transducing Interferon signals in epithelial tissues. We anticipate that Scribble may coordinate

HSC polarity to facilitate the proper response to inflammatory stress.

Results

Scribble deficient HSC are less responsive to IFN-I mediated HSC-activation and exhibit increased competitive repopulation following IFN-I stimulation

IFN-I is known to induce cell cycle arrest in hematopoietic progenitors populations (Verma et al., 2002) and we observed Wt mice treated with polyI:C (6 times, every other day at a dose of

10 mg/kg) exhibit hematologic deficiencies such as leukopenia, thrombocytopenia and anemia while hematopoietic specific Scribble deficient mice are insensitive to the short term anti- proliferative effects of IFN-I and retain normal blood count levels (Figure 4.1A). Paradoxically,

HSC from mice treated with IFN-I rapidly exit their quiescent state and enter the cell cycle depending on direct IFNAR signaling and activation of the signal transducer and activation of

STAT-1 and AKT (Essers et al., 2009; Platanias, 2005). Our preliminary data highlight that deficiency of Scribble in HSC renders them insensitive to IFN-I as they retain cellular quiescence in vivo (G0:44±14 vs 60±5.7% cellular quiescence in Wt and Scribble deficient HSC after poly I:C treatment respectively, p<0.05) (Figure 4.1B-C). In concordance with a loss of quiescence and an acquired entry into G1/S-G2-M phases of the cell cycle following IFN-I stimulation, HSC increase their surface expression of Sca-1 receptor (Essers et al., 2009) (Figure 4.1D). Consistent with the lack of cell cycle entry post IFN-I insult observed in the Scribble deficient HSC, we observed no statistically significant increase in surface Sca-1 expression between Scribble deficient HSC and Scribble deficient HSC treated with poly I:C (Figure 4.1D). Given that Scribble deficient HSC are less responsive to the acute effects of IFN-I, they are as a result functionally more fit and have increased reconstitution ability during serial transplantation (Figure 4.1E-F). PB chimerism displays a significant increase throughout secondary recipients and BM HSC display a 33% increase in chimera following secondary reconstitution (Figure 4.1F). Based on these data,

we hypothesize that the ME IFN-I induced HSC proliferative signaling program is mediated through Scribble.

Scribble deficient HSC mount an appropriate Stat1 signaling response following IFN-I stimulation

To test whether Scribble is involved in mediating the IFN-I driven STAT-1 response

(shown to be necessary for IFN-I driven cell cycle entry of HSC) we measured STAT-1 activity by its phosphorylation status, nuclear translocation and transcriptional target mRNA expression using an inducible Rosa26Cre;ScribbleΔ/Δ mouse model with or without poly I:C administration

(Figure 4.2A). Both Wt and Scribble deficient HSC harvested from mice that were treated with

Poly I:C displayed a similar 50% increase in STAT-1 activation compared to HSC harvested from mice that were untreated, evidenced by the right shifted population of STAT-1 phosphorylation

(Figure 4.2B). Since STAT-1 phosphorylation and activation permits its nuclear translocation, we subsequently examined STAT-1 nuclear localization. Immunofluorescence for total STAT-1 expression reveals a robust nuclear redistribution of STAT-1 in both Wt and Scribble deficient

HSC treated with Poly I:C (Figure 4.2C). Still, similar with STAT-1 phosphorylation, no differences in nuclear STAT-1 intensity were detected among Wt and Scribble deficient HSC post poly I:C

(Figure 4.2C-D). For completion, we measured IFN-I mediated response via expression of bona fide STAT-1 transcriptional targets (Essers et al., 2009; Walter et al., 2015) in HSC by RNA-seq and validated by qRT-PCR analysis. The majority of IFN-I mediated STAT-1 targets (oasl1, stat1 and pten) were unaffected by Scribble deficiency (Figure 4.2E-F). Interestingly, cxcl10 levels were significantly dampened in our transcriptomic analysis between Wt and Scribble deficient HSC after Poly I:C (Figure 4.2E). We validated this decreased expression of cxcl10 by qRT-PCR analysis (Figure 4.2F). Taken together, Scribble deficient HSC IFN-I insensitivity cannot be mediated through the obvious IFNAR-STAT-1 response as we detect no differences in STAT-1 phosphorylation, nuclear translocation and transcriptional activity, with the exception of cxcl10.

Scribble deficient HSC display lower Akt activity and Sca-1 membrane expression

HSC from mice treated with IFN-I rapidly exit their quiescent state and enter the cell cycle depending on direct IFNAR signaling and activation of STAT-1, AKT and upregulation of Sca-1

(Essers et al., 2009). Since we established STAT-1 effector response to IFN-I does not contribute to the Scribble deficient phenotype, we decided to investigate the role either proliferation mediator, Akt or Sca-1, has in driving IFN-I induced HSC activation. Akt phosphorylation has also been shown to be essential to regulate the switch between quiescence and proliferation in HSC

(Pietras et al., 2011) and there is a peak in Akt phosphorylation 3 days post IFN-I stimulation

(Pietras et al., 2014). We observed a dramatic upregulation of Akt protein expression (around 15 fold) in both poly IC treated hematopoietic progenitor cells. Given that, Pten transcriptional response to IFN-I was appropriately upregulated (Figure 4.2E-F) and Scribble has been shown to interact with the Phlpp1 phosphatase (Li et al., 2011), we thought it would be more likely that

Akt would be differentially modulated through the Phlpp1 residue. Indeed, we observed a slight decrease in Akt activity within the Scribble deficient cells measured by phosphorylation at serine

473 (Figure 4.2G). Sca-1 represents another vital regulator of cellular proliferation response to

IFN-I as the loss of Sca-1 abrogates IFN-I induced proliferation of HSC (Essers et al., 2009; Walter et al., 2015). Ly6a is appropriately upregulated in response to IFN-I with or without Scribble protein expression. However, Sca-1 surface expression is significantly diminished in Scribble deficient

HSC following acute IFN-I stimulation (Figure 4.1D).

Discussion

Our results highlight that Scribble deficient HSC remained less responsive to IFN-I mediated HSC activation. As a result, Scribble deficient HSC were functionally more fit evidenced by an increase in competitive repopulation abilities following acute IFN-I stimulation. Despite the

dominant role of Stat1 in influencing HSC activation following IFN-I stimulation, Stat1 effector signaling appropriately responded to IFN-I regardless of Scribble status. Interestingly, we observed lower levels of Akt phosphorylation at the Phlpp1 regulated serine residue 473.

The lack of IFN-I mediated HSC activation observed in Scribble deficient HSC could be partially explained from the lower induction of Akt phosphorylation and activity. Akt is a very well characterized proliferation driver and its activity is tightly modulated through the phosphorylation and dephosphorylation events. Two very prominent phosphatases, Pten and Phlpp1, have the capacity to inactivate Akt by dephosphorylating the enzyme at conserved residues threonine 308 and serine 473, respectively (Bozulic and Hemmings, 2009). Scribble has been shown to bind

Phlpp1 and coordinate its membrane localization to facilitate negative regulation of Akt (Li et al.,

2011). However, the opposite is observed in our study, Scribble deficient HSC display decreased phosphorylation of Akt 473 in response to IFN-I. Further experimentation will be required to determine if Scribble directly controls Akt 473 phosphorylation through scaffolding of Phlpp1 in

HSC.

Interestingly, Scribble deficient HSC displayed a significant loss of Sca-1 surface expression, another prominent proliferation regulator in HSC biology. Sca-1 surface expression controls HSC quiescence and self-renewal potential (Morcos et al., 2017). Sca-1 also represents a vital component for the cellular responses to IFN-I as the loss of Sca-1 abrogates IFN-I induced proliferation of HSC (Essers et al., 2009; Walter et al., 2015). Ly6a, a Stat1 transcriptional target, is appropriately upregulated in response to IFN-I with or without Scribble protein expression.

However, Sca-1 surface expression is significantly diminished in Scribble deficient HSC following acute IFN-I stimulation. Work on LET-413 (C. elegans homologue of Scribble) provides evidence that Scribble organizes protein trafficking, coordinating Rab5 effector to regulate activation of

Rab10 and thus promoting endocytic recycling (Liu et al., 2018). We are extremely interested and actively pursuing the prospect that Scribble can regulate Sca-1 surface expression in HSC through endosomal trafficking mechanisms.

HSC lacking the IFNα/β receptor (IFNAR), Stat1 or Sca-1 are insensitive to IFN-I stimulation, demonstrating that IFNAR, STAT1 and Sca-1 are critical to mediate IFN-I induced

HSC proliferation (Essers et al., 2009). As both Akt and Sca-1 expression are crucial regulators of HSC proliferation in response to IFN-I (Essers et al., 2009; Pietras et al., 2011; Walter et al.,

2015), we anticipate that Scribble coordinates IFN-I mediated HSC activation by controlling

Phlpp1 localization and subsequent Akt activity, as well as, regulating endosomal membrane trafficking of Sca-1 protein (Figure 4.3). Elucidation of the precise molecular mechanisms through which Scribble controls IFN-I mediated HSC activation could prove useful in developing therapeutic agents that would presumably dampen inflammation driven HSC exhaustion in patients treated with IFN-I, as well as alleviate the involvement of inflammation in HSC aging and disease progression.

Viral proteins evolve by antagonizing the interferon signaling pathways elicited by the host to eradicate viral infection (Alcami et al., 2000; Colamonici et al., 1995; Ronco et al., 1998). The

Human Papillomavirus (HPV) produces the 16E6 oncoprotein that binds selectively to cellular

Interferon regulatory factor 3 to thwart the host antiviral response (Ronco et al., 1998).

Interestingly, human Scribble was identified as a specific target, via PDZ protein interactions, for the HPV E6 oncoprotein (Nakagawa and Huibregtse, 2000). This targeted depletion of Scribble in a subset of cervical cancers supports the possible function of Scribble in transducing Interferon signals and is likely to be regulated by other viruses known to infect and damage HSC like parvovirus B19 among others (Morinet et al., 2011).

Material and Methods

Animals. Exons 2-8 of the Wt Scribble locus were conditionally targeted to create Scribble flox/flox alleles. LoxP sites were introduced into the introns 1 and 8 flanking Scribble exons 2 through 8 in mouse embryonic stem cells, using a selectable neo cassette flanked by frt sites. Targeted stem cells were injected into blastocysts of C57BL/6J mice to obtain chimeric floxed mice. After germline transmission, the mice were crossed to C57BL/6J mice expressing flp recombinase to remove the neo cassette. Progeny containing floxed Scribble alleles lacking the neo cassette

(Scribbleflox/flox) were further backcrossed to C57BL/6J mice. Construction of the targeting vector, generation and injection of targeted stem cells and subsequent generation of chimeric mice were performed by inGenious Targeting Laboratory (New York, United States of America).

The Scribble flox/flox mice were also crossed to an inducible Rosa26Cre-ERTi2Tg/- background for inducible ubiquitous deletion. Cre-mediated recombination causes a frame shift and early stop of

Scribble protein translation. Induction of Cre recombinase expression in inducible transgenic animals was achieved by by administration of 1mg tamoxifen (10mg/mL) intraperitoneal twice a day for 4 days for the Rosa26Cre-ERTi2Tg/- animals. Acute IFN-I stimulation in transgenic animals was achieved by injections of polyinositide/cytidine (polyI:C) at an intraperitoneal dose of 10 mg/kg every other day for the time course described in the text.

Littermate mice from the same breeding pair were used in all experiments. All mouse strains were maintained at an Association for Assessment and Accreditation of Laboratory Animal Care accredited, specific-pathogen–free animal facility at Cincinnati Children’s Hospital Medical

Research Foundation, Cincinnati, under an Institutional Animal Care and Use Committee approved protocol. All mice were between 6 and 12 wk of age at the time of experimentation.

Mice genotypes were determined by PCR analysis. Sequences of genotyping primers are available upon request.

Fluorescence activated cell sorting (FACS) and Immunophenotypic analysis of HSC/P.

Bone Marrow cells were stained using a mixture of biotin-conjugated monoclonal anti-mouse lineage antibodies against CD45R (B220, Clone RA3-6B2), Gr-1 (Ly6G, Clone RB6-8C5), CD4

(Clone RM4-5), CD8a (Ly-2, Clone 53–6.7), Mac-1 (CD11b, CloneM1/70), CD3ε (Clone 145–

2C11), and TER119 (Ly-76) (all from BD). In a subsequent labeling step, the cells were incubated with a combination of streptavidin-allophycocyanin (APC)-Cy7 (BD), phycoerythrin (PE) Cy7 anti- mouse Sca-1 (Ly6A/E, clone D7; BD), APC anti-mouse CD117 (c-kit, Clone 2B8; BD), PerCP- cy5.5 anti-mouse CD16/32 (Clone 2.4G2;BD) FITC anti-mouse CD48 (Clone HM481; BD) or

BV605 anti-mouse CD48 (Clone HM481;BD), eFluor 450 anti-mouse CD34 (Clone RAM34; eBioscience), PE anti-mouse CD135 (FLT3, CloneA2F10.1) or PE anti-mouse CD150 (Clone mShad150; eBioscience) antibodies. Analysis of bone marrow nuclear cells harvested from all genotypes were immunophenotypically defined by differential expression of cell surface antigens:

Lin- C-kit+ Sca-1+/- CD135+/- CD34+/- CD48+/- CD150+/- CD16/32+/-. Active Stat1 was analyzed in hematopoietic progenitors using PE conjugated Rabbit anti-P-Stat1 (Y701) (Cell Signaling

Technologies, 5806).

PyroninY (0.25 mg/mL). Verapamil (100 µM) was added during staining and analysis to prevent extrusion of Hoechst 33342.

Confocal immunofluorescence microscopy analysis. HSC sorted (using BD FACSAria II) from all geneotypes were seeded onto fibronectin (RetroNectin catalog T100B, TAKARA BIO

INC.) coated glass chamber slides in Stemspan medium (Stem Cell Technologies, 09600) containing mouse stem cell factor (100ng/mL) and Thrombopoietin (100 ng/mL). Cells were cultured overnight (10-12 hours) at 37 degree Celsius and then fixed in 4% paraformaldehyde for

20 minutes at 4°C, permeabilized with 0.1% Triton X-100 (catalog T9284, Sigma-Aldrich) for 10 minutes at room temperature, and blocked with 5% Donkey serum in PBS for one hour. The slides were stained with primary antibodies in 5% Donkey Serum at 4°C overnight: Rabbit anti-Stat1

(Cell Signaling Technologies, 14994). The cells were washed with PBS twice and then labeled with secondary antibodies (from Invitrogen): Donkey anti–rabbit Alexa Fluor 488 (catalog A21206) at 1:500 v/v concentration for 2 hours at room temperature. Cells were washed with PBS and then slides were mounted using Prolong Gold Antifade mounting media (Thermo Fisher Scientific,

P36935) containing DAPI. Stained cells were imaged with a Nikon Ti-E Inverted A1R Confocal

Microscope with GaAsp PMTs, Resonant Scanner, Piezo Z-Drive, Andor iXon 888 EMCCD

Widefield Camera. Images were analyzed and processed using NIS-Elements and Adobe

Photoshop v7.

Serial competitive repopulation transplantation. Equal amounts of BM from CD45.2+ Wt and

ScribbleΔ/Δ mice were mixed with congenic CD45.1+ B6.SJLPtprcaPep3b/BoyJ competitor cells and were transplanted at a 1:1 ratio into lethally irradiated B6.SJLPtprcaPep3b/BoyJ recipients. Peripheral blood chimera (measured by CD45.2+ leukocytes) of primary, secondary and tertiary recipients were measured every 4 weeks and absolute blood counts were recorded. Peripheral blood FACS analysis of CD45.2 chimera and lineage reconstitution was performed by staining peripheral blood cells using monoclonal anti-mouse lineage antibodies against CD45.2 (Clone 104), CD45.1

(Clone A20), CD45R (B220, Clone RA3-6B2), CD11b (M1/70) and CD3ε (Clone 145-2C11). After

16 weeks of reconstitution, recipients were sacrificed and PB, BM and spleen were analyzed for total CD45.2 chimera, lineage reconstitution/ absolute lineage positive cellularity and HSC/P

analysis was performed on BM and spleen. BM cells were pooled from each recipient for each genotype and serially transplanted into secondary recipients.

Weastern Blot Analysis. Hematopoietic stem and progenitor cells were sorted and lysed using

RIPA (Cell Signaling Technologies, 9806). Lysates were collected and samples were prepared for gel electrophoresis by denaturing proteins with SDS containing β-mercapto-ethanol and boiled for 10 min. Samples were run at 100Volts for 1 hour through a 4-12% gel and subsequently transferred at 75Volts for 2 hours to a PVDF membrane (Millipore, IPVH00010) followed by blocking with 5% milk and primary antibody incubations overnight at 4 degree: Mouse anti-Akt

(cell signaling technologies, 2967) Rabbit anti-P-Akt(S473) (cell signaling technologies, 9018).

After washing with TBS-T, an anti-Rabbit Secondary HRP antibody (Cell Signaling Technologies,

7074) and an anti-Mouse Secondary HRP antibody (Cell Signaling Technologies, 7076) were incubated for 1.5 hours at room temperature. Membranes were washed again with TBS-T and developed using LumiGLO peroxide reagent (Cell Signaling Technologies, 7003S), autoradiography film (Danville Scientific, XC59X) and a developer.

Transcriptome and Bioinformatics Analysis. Total RNA was extracted from sorted HSC derived from Rosa26Cre;Wt or ScribbleΔ/Δ mice one day following polyinositide/cytidine (polyI:C) administration and acute IFN-I stimulation using RNeasy Mini Kit (QIAGEN). RNA quality and concentration were measured by Bioanalyzer 2100 using the RNA 6000 Nano Assay. The initial amplification step was performed with the Ovation RNA-Seq System v2 (Tecan Genomics) and the cDNA concentration and size were measured using the Qubit dsDNA BR assay and a DNA

1000 Chip, respectively. The subsequent libraries were prepared with the Nextera XT DNA

Sample Preparation kit (Illumina Technologies). Briefly, 1ng of cDNA was suspended in Tagment

DNA Buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (Amplicon Tagment Mix), incubating at 55C for 10 min. NT Buffer was then added to neutralize the samples. Libraries were prepared by PCR with the Nextera PCR Master

Mix, and 2 Nextera Indexes (N7XX, and N5XX) according to the following program: one cycle of

72C for 3min, one cycle of 98C for 30s, 12 cycles of 95C for 10s, 55C for 30s, and 72C for 1min, and one cycle of 72C for 5min. The purified cDNA was captured on an Illumina flow cell for cluster generation and Libraries were sequenced on the Illumina HiSeq2500 following the manufacturer's protocol. The concentration of the pool was optimized to acquire at least 25-30 million reads per sample using Paired-End Reads with a read length of 75 bps. Reads were aligned with TopHat software, using hg19 as the reference genome and Reads Per Kilobase of transcript per Million mapped reads (RPKM) as output. RPKM were log2-transformed and baselined to the median expression of the average of each class of samples. Analysis of differential gene expression and gene ontology (GO) pathway analyses using Altanalyze software (developed by Cincinnati

Children’s Research Foundation). cDNA was kept from initial pool for subsequent qRT-PCR validation of RNA-seq hits.

Statistical analysis.

Data are presented as average ± standard deviation. Comparisons were performed with Student t test, Chi-squared test and one-way or two-way ANOVA when required. Statistical significance levels were established at 5%, 1% and 0.1%.

Acknowledgements

This study was supported (in part) by the founding from the National Institutes of Health (F31HL132468) and the University of Cincinnati (Albert J Ryan Foundation and Cardell Fellowship for Excellence in Graduate Research), awarded to MJA.

Author contributions

MJA, AMW, BB performed experiments. MJA and JAC analyzed data and wrote the manuscript. MJA and JAC designed experiments. JAC led the group and supervised MJA.

Disclosure of conflicts of interest

The authors declare no relevant conflicts of interest

Figures and Legends

Figure 4.1. Scribble deficient HSC are less responsive to IFN-I mediated HSC activation.

(A) Peripheral blood counts following acute IFN-I stimulation in primary Vav1Cre;Scribblefl/fl animals. WBC: White blood cell, ANE: Absolute neutrophil count.*p<0.05, ***p<0.001. (B) Acute

IFN-I treatment regimen prior to HSC activation analysis. (C) Cell cycle analysis between HSC isolated from Wt and Scribble deficient with and without IFN-I stimulation. *p<0.05. (D) Sca-1 surface expression MFI in HSC isolated from Wt and Scribble deficient mice treated with and without IFN-I. **p<0.01. (E) Peripheral blood chimerism (CD45.2+ reconstitution) during competitive repopulation analysis of Wt and Scribble deficient HSC treated with IFN-I prior to transplant. *p<0.05, **p<0.01, ***p<0.001. (F) CD45.2+ HSC percentage from the BM following secondary reconstitution. *p<0.05.

Figure 4.2. Stat1 activation and transcriptional impact in response to IFN-I remains relatively unchanged. (A) Schematic representing a ubiquitous Rosa26Cre;Scribblefl/fl animal model where excision of the Scribble gene is accomplished using tamoxifen one week prior to acute IFN-I stimulation and HSC activation analysis. (B) Flow cytometry measuring active

(phosphorylated) Stat1 MFI in LSK BM progenitors and HSC. (C) Immunofluorescence depicting

Stat1 nuclear translocation in HSC treated with IFN-I, Scale bar 5µm. (D) Quantification of nuclear

Stat1 MFI in Wt and Scribble deficient HSC post IFN-I stimulation, p=ns. (E) RNA sequencing analysis of bone a fide Stat1 transcriptional targets, **p<0.01. (F) Stat1 transcriptional target validation using quantitative RT-PCR on RNA isolated from Wt and Scribble deficient HSC with or without IFN-I stimulation. ***p<0.001. (G) Western blot analysis of Akt expression and activation between Wt and Scribble deficient hematopoietic stem and progenitor cells with or without IFN-I stimulation.

Figure 4.3. Potential mechanism underlying Scribble deficient HSC activation following

IFN-I stimulation. Stat1 signaling remains unaffected in Scribble deficient HSC following IFN-I stimulation while we detected less Akt activation and Sca-1 surface expression, both events presumably could hinder proliferation and survival pathways.

Chapter 5: Discussion, Implications and Future Directions

We identified that the polarization of Scribble in HSC coordinates the organization and activation of the Hippo signaling pathway via PDZ domain mediated interactions, associates with

Cdc42 to modulate its activity, and regulates HSC divisional fate. Our data provide the first genetic demonstration that Hippo-regulated Yap1 controls HSC quiescence and fitness while the combined Yap1/Taz/Scribble complex controls Cdc42 activity and proper HSC fate decisions

(Figure 5.1).

Figure 5.1. Proper HSC self-renewal and asymmetric divisions are coordinated by a polarized Scribble/Yap1-Taz/Cdc42 complex. Schematic depicting the specific consequences pertaining to HSC fate resulting from the genetic ablation of Cdc42, Yap1 and Taz or Scribble.

Loss of Cdc42 protein expression has been reported to produce hyperproliferation of hematopoietic stem and progenitor cells followed by exhaustion of functional HSC as mice eventually succumb to bone marrow failure (Yang et al., 2007). A similar loss of HSC fitness is observed when we delete Taz and Yap1 in HSC and subject them to physiological stress such as inflammation, transplantation or chemotherapy. Given that the genetic deletion of Yap1 and Taz

results in decreased Cdc42-GTP activity, Cdc42 is likely to be the more downstream effector in our studies, and thus a more severe phenotype is presented upon its loss compared to the loss of Yap1 and Taz. Interestingly, the genetic loss of Scribble and its associated lack of polarity between Yap1, Taz and Cdc42, provides the system with increased HSC fitness as self-renewing divisions become preferential. The loss of all three components, Scribble, Yap1 and Taz, results in a neutralizing effect between the two independent phenotypes and is associated with Rac1 upregulation and activation. Specifically, the additional deletion of Scribble in Yap1/Taz deficient

HSC rescues cell cycle quiescence and survival in response to 5-FU, compared to the double deficiency of Yap1 and Taz. Small GTPases are known to influence HSC activity (Cancelas et al.,

2005; Florian et al., 2012; Hinge et al., 2017) and our data contribute to the robustness and complexity of the cross-talk between Rho, Rac and Cdc42.

The fact that the canonical signaling pathway downstream of Cdc42, aPKC/Par3/Par6, does not impair hematopoiesis (Sengupta et al., 2011) suggests that a mechanism of Yap1 regulation via direct phosphorylation by PKCζ (Llado et al., 2015) is dispensable in HSC, and an alternative pathway may control HSC fitness. The critical role of Yap1 and Taz in regulating biomechanical properties of cells and subsequent mechanotransduction (Dupont et al., 2011) may provide insights towards an uncharacterized functional requirement for Yap1 and Taz in HSC activity. It has become increasingly evident that intrinsic and extrinsic mechanical properties

(resistance to deformation/ elasticity or flow/ viscosity) in response to an applied force, regulate cellular behaviors, such as cell morphology, adhesion, migration and trafficking. Interestingly,

HSC cultured on matrices of different elasticity confirmed that self-renewal and differentiation of these stem cells are mechanosensitive (Holst et al., 2010; Ni et al., 2019; Shin et al., 2014).

Recently, Septin1, a GTP-binding protein downstream of the protein phosphatase Ptpn21, has been shown to be essential for maintaining cortical cytoskeleton integrity, stability and tension within HSC (Ni et al., 2019). Proper maintenance of these mechanical properties has proven to be indispensable in regulating HSC niche retention, quiescence and overall regenerative capacity.

Along these lines, biomechanical force induced Rho-GTPase mediated activation of Yap1 signaling has been shown to instruct primitive hematopoietic stem cell specification, production, and maturation in vivo (Lundin et al., 2020). While de novo production of HSPC provides significant therapeutic value, current in vitro approaches cannot efficiently generate multipotent long-lived HSPC. Presuming this reflects a lack of extrinsic cues controlling normal developmental specification of HSPC from hemogenic endothelium in the ventral dorsal aorta, Lundin et al engineered a human dorsal aorta-on-a-chip platform that identified Yap1 as an important regulator of HSPC formation in vitro. Likewise, biomechanical force resulting from blood shear stress, stimulated Rho-GTPase activity that lead to Yap1 driven endothelial-to-hematopoietic transition in vivo (Lundin et al., 2020). These findings reveal a novel Rho-mediated Yap1 mechanotransduction mechanism on hemogenic endothelium fate relevant for HSC specification.

We provide evidence that the complete loss of Yap1/Taz results in non-viable pups when breeding hematopoietic-specific Vav1CreTg/-;Yap1f/f;Tazf/f mice, whereas the incomplete loss of one to three alleles (between both Yap1 and Taz) did not impair the expected Mendelian inheritance ratios (Supplemental Figure 3.1A). These data suggested that the complete loss of

Yap1/Taz severely impairs hematopoietic development resulting in fetal death. Since circulation onset begins around e8.25-e8.5 post coitum (Dzierzak and Speck, 2008) and our Yap1/Taz null fetuses exhibited regression prior to e10.5, the newly found role of Yap1 on HSC specification might provide mechanistic insights for the observed embryonic lethality in our Yap1/Taz null embryos. Further experimentation must be conducted to identify which Rho-GTPase is responsible for integrating these biomechanical signals during embryonic development to regulate Yap1 activity and HSC specification.

Yap1 was believed to be dispensable in definitive hematopoiesis (Donato et al., 2018;

Jansson and Larsson, 2012), however we provide evidence for a functional complex containing

Yap1, Scribble, p-Lats and Cdc42/Cdc42-GTP in dividing HSC that controls quiescence, fate determination and overall fitness. Even though our studies did not directly investigate a role for

Yap1 in HSC mechanotransduction, it would interesting to hypothesize that our complex is working in concert with upstream mechanosensitive effectors to control HSC fate.

Cdc42 allocation has been mathematically defined to predict asymmetric division potential

(Florian et al., 2018). We have confirmed that HSC frequently display an asymmetric distribution of the proliferation and fate determinant Cdc42 (Florian et al., 2012; Florian et al., 2013).

Additionally, we provide evidence that Scribble introduces Hippo proteins like Lats1 and Yap1/Taz to this polarized complex to modulate Cdc42 activity. Asymmetric segregation of molecular fate determinants has been proposed as a potential mechanism for asymmetric stem cell divisions among both murine and human HSPC (Beckmann et al., 2007; Brummendorf et al., 1998; Cheng et al., 2019; Ito et al., 2012; Wu et al., 2007). In addition to Cdc42, other fate determinants like

AP2A, Tie2, CD63, Myc, Numb, active mitochondria and lysosomal machinery (Beckmann et al.,

2007; Cheng et al., 2019; Hinge et al., 2020; Ito et al., 2012; Loeffler et al., 2019; Ting et al., 2012;

Vannini et al., 2019; Wu et al., 2007; Zimdahl et al., 2014) have been suggested to be asymmetrically inherited during HSC divisions. However, with the exception of mitochondrial inheritance, no functional relevance for such asymmetrically inherited fate determinants has yet been demonstrated in HSC. Given that HSC show significant co-polarization between Scribble,

Yap1 and Cdc42, and that Scribble deficiency reduces the proportion of asymmetric cell divisions among HSC (Figure 5.2), we are interested in following up these studies to investigate whether or not Scribble polarization and/ or Yap1 distribution would be a useful indicator of asymmetric division potential, akin to Cdc42 (Florian et al., Plos Biology 2018). HSC predominantly display co-polarization between Scribble, Yap1 and Cdc42 (represented by 75% of HSC). Scribble deficiency decreases the preponderance of co-polarization events (only observed in roughly 30% of HSC) and shifts the prevalence towards non-polarized (non-polar-35%, and even non- interacting-35%) cells, which functionally correlates with an increased proportion of symmetric self-renewal divisions at the expense of fewer asymmetric divisions.

Figure 5.2. HSC that display increased co-polarization between Scribble, Yap1 and Cdc42, harbor greater asymmetric division potential. Cartoon depicting Scribble interactions with

Yap1 and Cdc42 in Wt or Scribble deficient HSC. Interactions are defined as interacting and polarized (co-polarization), interacting but apolar (non-polar), or non-interacting events that are also apolar (non-interacting).

Along these lines, Scribble has been shown to be asymmetrically inherited during muscle stem cell divisions (Ono et al., 2015). Scribble is asymmetrically distributed in dividing satellite cells (skeletal muscle stem cells), with robust accumulation in cells committed to myogenic differentiation (Ono et al., 2015). In fact, the reduction in asymmetric division potential observed when Scribble was deleted in HSC was more dramatic in functional assays due to a selective loss of the asymmetric division daughter cells in clonal outgrowth cultures, implying that Scribble may be required for proper differentiation of progenitors after the primary HSC division. To appropriately address this notion, we would need to cross our Scribble deficient mice with a fluorescent reporter to image Scribble inheritance in individual daughter cells and assess the resulting functionality.

An argument can also be made for the molecular dependence of Yap1 and Taz allocation during HSC divisions. We provide evidence that Yap1 and Taz are essential positive regulators

of HSC self-renewal (Figure 3.1) and that apolar distribution of Yap1 and Taz (consequence of

Scribble deficiency) increases symmetric self-renewal potential (Figure 3.4 and 3.5). It is likely that Scribble expression may force asymmetric division potential by polarizing Yap1 and Taz, thus controlling their asymmetric inheritance in daughter HSC. However, in the absence of Scribble, there is an increased frequency of apolar HSC in regards to Yap1 and Taz distribution. Such a decrease in Yap1 and Taz asymmetry might influence the overall inheritance of Yap1/ Taz, and subsequently shift HSC divisions to favor symmetric self-renewal (Figure 5.2).

Historically, to demonstrate asymmetric cell division of an HSC, researchers have relied on one of two approaches: either analysis of a) asymmetric daughter-cell functional output after division or b) on the asymmetric inheritance of fate determinants during division or immediately following division (Ito et al., 2016; Pham et al., 2014; Ting et al., 2012; Wu et al., 2007; Zimdahl et al., 2014). Until recently, these two approaches could not be combined due to experimental technicalities that prevented analysis of functional daughter cell output with fate determinants observed in fixed samples. The generation of reporter systems (proteins fused with fluorescent tags) combined with live cell imaging have made coupling these two approaches a possibility and will tremendously expand the coming discoveries on HSC fate determination (Christodoulou et al., 2020; Hinge et al., 2020; Loeffler et al., 2019).

Recently, the cellular degradative machinery including lysosomes, autophagosomes, mitophagosomes and the protein Numb were described to be asymmetrically inherited together in HSC daughter cells in vitro (Loeffler et al., 2019). In this report, Numb is merely a reporter for asymmetric inheritance as no functional requirement was described for its inheritance. However, the asymmetric inheritance of the autophagosomal and/or lysosomal degradative machinery is predicted to regulate mitochondrial clearance and autophagy to coordinate metabolic and translational activation among HSC daughter cells. The functional dependence of the inherited machinery (organelles and proteins) lends further credence towards the notion that asymmetric partitioning of proteins prior to division can in fact influence HSC fate decisions and function. The

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fact that the HSC pool is a highly heterogeneous group of cells, with different divisional history and kinetic levels at any given time, makes it crucial to apply methods to trace HSC fate in future experiments. Advancements are being made in the analysis of fate determinant allocation in vivo using novel transgenic fluorescent reporter approaches and using lentiviral transduction of barcodes that identify the clonal contribution of each independent HSC.

Consistent with the notion of HSC heterogeneity, we observe that Scribble is polarized in

80% of SLAM HSC (potentially asymmetrically inherited upon successful division), leaving 20% of immunophenotypically defined SLAM HSC that are not asymmetric or polarized with respect to

Scribble expression (Figure 5.3). Notably, most primitive HSC (HSC with durable self-renewal,

Figure 5.3. Scribble protein localization is apolar in more primitive durable self-renewing

HSC. (A) Immunofluorescence depicting Scribble protein localization in Wt durable self-renewing

- - + - + - HSC (immunophenotypicaly defined as LSK CD34 Flt3 CD150 CD48 EPCR CD49b ) and

+ - SLAM HSC (immunophenotypically defined as LSK CD150 CD48 ). Scale bar is 5 µm. (B)

Quantification for the frequency of HSC with Scribble polarization.

(Kent et al., 2009)), display an apolar distribution of Scribble (Figure 5.3). This might imply that

Scribble polarization is differential in certain subsects of HSC. Either these HSC have true independent functional capabilities from the bulk SLAM HSC population (further proof of a

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heterogeneous group), or simply they represent a transitional state, that is to say, that apolar

Scribble distribution is only seen in dormant HSC, however, upon activation, Scribble is rapidly reorganized and redistributed to the polarized form we observe in roughly 80% of the bulk SLAM

HSC. The fact that this distribution discrepancy corresponds with a specific sub-population of

HSC shown to possess more durable self-renewal potential (Kent et al., 2009), strengthens the argument that apolar distribution of Scribble may confer a higher order of stemness.

Importantly, the phenotype exhibited by Scribble deficient HSC mirrors the effects observed when the Scribble complex partner, Lgl1, is conditionally deleted in HSC (Heidel et al.,

2013). In both scenarios, the deficiency of each respective Scribble complex gene lead to a loss of HSC quiescence and an acquired increase in the reconstitution abilities, thus implying that HSC biology is greatly dependent upon the redundancy and cooperativity between Scribble complex partners. However, a recent report claims that the loss of Scribble results in the opposite, a decreased competitive reconstitution ability using an interferon-mediated hematopoietic inducible

Mx1Cre model (Mohr et al., 2018). Coupling the nature of IFN-I on HSC activation with the fact that we provide evidence describing Scribble deficient HSC as less sensitive and responsive to

IFN-I, the reported loss of competitive advantage in Scribble deficient HSC could be conceivable for this context. Even though, no mechanism has been provided to explain the apparent loss of hematopoietic reconstitution, this report has utilized mass spectroscopy data in hematopoietic cell lines and identified regulators of the cytoskeleton network and GTPase activity as putative

Scribble interacting partners. Likewise, Rho GTPase signaling and activity were prominent differentially regulated gene clusters in our transcriptomic analysis of Scribble deficient HSC. We provide the first mechanistic evidence that Scribble spatially coordinates interactions between

Hippo signaling pathway effectors and the Rho GTPases, Cdc42 and Rac1, to control HSC polarization, fate and function. Collectively, the data presented throughout this dissertation strongly support a role for Scribble in HSC quiescence, signaling and fate determination.

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Chapter 6: Conclusions

Conclusions regarding the role of Scribble-mediated HSC polarity and its contribution in stem cell division and fate:

1. Deficiency of Yap1 and its paralogue Taz results in loss of HSC quiescence and overall fitness. 2. Gain of nuclear Yap1 activity does not provide any significant impact on HSC lending further credence for the existence of a cytosolic function of Yap1 in HSC. 3. Scribble scaffolds cytosolic Hippo/Yap1 complex polarization and is required for Cdc42 activity. 4. Polarization of Yap1 can be restored in Scribble deficient HSC by forced expression of either Scribble full length or its PDZ domain. 5. Loss of Scribble, and its associated Yap1/Cdc42 polarization, increases the prevalence of self-renewing divisions among HSC which manifests as increased HSC fitness. 6. Loss of Scribble, Yap1 and Taz collectively diminishes Cdc42 expression and activity. 7. Loss of Scribble restores HSC self-renewal of Yap/Taz deficient HSC and associates with increased Rac 1 activity.

Conclusions regarding Scribble-mediated HSC polarity and its regulation of the HSC stress- response to Interferon signaling:

1. Scribble deficient HSC are less sensitive to IFN-I induced HSC activation and exhibit increased competitive repopulation following IFN-I stimulation. 2. Scribble deficient HSC mount an appropriate Stat1 signaling response following IFN-I stimulation measured by its phosphorylation, nuclear translocation and activation of bona fide Stat1 transcriptional targets. 3. Scribble deficient HSC display lower Akt activity and Sca-1 membrane expression, two crucial regulators of IFN-I mediated HSC activation.

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Chapter 7: Impact and Relevance

In summary, the work presented in this dissertation identifies Scribble and Hippo-regulated cytosolic Yap1 and Taz as crucial regulators of HSC polarity and fate decisions in the context of transplantation, chemotherapy and inflammation.

Notably, this pathway (and protein complex) is amenable to genetic and/or pharmacological intervention for the ex vivo manipulation of transplantable HSC, aimed towards cell and gene therapy approaches.

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