ABSTRACT 5 PAPERS IN THIS THESIS 7 ABBREVIATIONS 8 INTRODUCTION 9 ORGANOGENESIS 9 THE LIVER 10 Early liver development 12 Mesenchymal-epithelial interactions during hepatic induction, morphogenesis and growth 13 THE HEMATOPOIETIC SYSTEM 15 Development of the hematopoietic system 17 Hematopoietic stem cell phenotype 18 The role of the microenvironment in HSC self-renewal 19 ES cells differentiated in vitro mimic early hematopoietic development 20 THE OLFACTORY SYSTEM 21 Organization and development of the olfactory epithelium 22 Development and differentiation of OSNs 23 Odorant receptors 25 OE zones 25 LIM-HD TFs AND REGULATION OF PATTERNING AND CELL SPECIFICATION 27 Lhx2 AND ORGANOGENESIS 29 AIMS 31

RESULTS AND DISCUSSION 32 Lhx2 AND LIVER DEVELOPMENT (paper I) 32 Lhx2 is expressed in hepatic mesenchyme 32 The mutant phenotype is due to defective mesenchyme 33 Possible mechanisms of Lhx2-mediated liver expansion 34 Lhx2 AND HEMATOPOIESIS (paper II) 36 Lhx2 immortalizes multipotent hematopoietic progenitor cells 36 Lhx2 may regulate HSC self-renewal in vivo 38 Dkk-1, A PUTATIVE Lhx2 REGULATED GENE IN THE HEMATOPOIETIC AND HEPATIC SYSTEM (paper III) 39 Dkk-1 alone cannot mediate HPC self-renewal 40 Dkk-1 acts via a Wnt/ß-catenin independent mechanism in the liver 41 Lhx2 AND THE OLFACTORY SYSTEM (paper IV) 43 Lhx2 is required for the expression of OR genes and other markers of mature OSNs 44 Accumulation of immature neurons in Lhx2-/- embryos 45 Lhx2 and regional specificity 45 Lhx2 – a putative regulator of OR gene expression 46 A role for Lhx2 in olfactory progenitor cells? 47 SUMMARY OF RESULTS AND DISCUSSION 48 CONCLUSIONS 49 ACKNOWLEDGEMENTS 50 REFERENCES 52 PAPERS I-IV

ABSTRACT

During embryonic development a variety of tissues and organs such as the lung, eye, and kidney are being formed. The generation of functional organs is regulated by reciprocal cell-cell interactions. Via the secretion of soluble molecules one type of cells affect the fate of their neighboring cells. A central issue in organogenesis is how a cell interprets such extrinsic signals and adopts a specific fate, and how the cell in response to this signal establishes reciprocal signaling. Transcription factors play a critical role in this process and my thesis focuses on the role of the LIM- homeodomain transcription factor Lhx2 in the development of three different organ systems, the liver, the hematopoietic system and the olfactory system. The liver is formed from endoderm of the ventral foregut and mesenchyme of the septum transversum (st) and its development depends upon signaling interactions between these two tissues. As the liver becomes a distinct organ it is colonized by hematopoietic cells and serves as hematopoietic organ until birth. The fetal liver provides a microenvironment that supports the expansion of the entire hematopoietic system (HS) including the hematopoietic stem cells (HSCs). Liver development in Lhx2-/- embryos is disrupted leading to a lethal anemia due to insufficient support of hematopoiesis. To further investigate the role of Lhx2 in liver development I analyzed gene expression from the Lhx2 locus during liver development in wild-type and Lhx2-/- mice. Lhx2 is expressed in the liver associated st mesenchymal cells that become integrated in the liver and contribute to a subpopulation of hepatic stellate cells in adult liver. Lhx2 is not required for the formation of these mesenchymal cells, suggesting that the phenotype in Lhx2-/- livers is due to the presence of defective mesenchymal cells. The putative role of Lhx2 in the expansion of the HS was examined by introducing Lhx2 cDNA into embryonic stem cells differentiated in vitro. This approach allowed for the generation of immortalized multipotent hematopoietic progenitor cell (HPC) lines that share many characteristics with normal HSCs. The Lhx2-dependent generation of HSC-like cell lines suggests that Lhx2 plays a role in the maintenance and/or expansion of the HS. To isolate genes putatively linked to Lhx2 function, genes differentially expressed in the HPC lines were isolated using a cDNA subtraction approach. This allowed for the identification of a few genes putatively linked to

Lhx2 function, as well as several stem cell-specific genes. The antagonist of Wnt signalling, Dickkopf-1 (Dkk-1), was identified in the former group of genes as it showed a similar expression pattern in the fetal liver, as that of Lhx2 and expression of Dkk-1 in fetal liver and in HPC lines appeared to be regulated by Lhx2. This suggests that Dkk-1 plays a role in liver development and/or HSC physiology during embryonic development. During development of the olfactory epithelium (OE) neuronal progenitors differentiate into mature olfactory sensory neurons (OSNs) that are individually specified into over a thousand different subpopulations, each expressing a unique odorant receptor (OR) gene. The expression of Lhx2 in olfactory neurons suggested a potential role for Lhx2 in the development of OSNs. To address this, OE from Lhx2-/- and wild-type mice was compared. In the absence of functional Lhx2 neuronal differentiation was arrested prior to onset of OR expression. Lhx2 is thus required for the development of OSN progenitors into functional, individually specified OSNs. Thus, Lhx2 trigger a variety of cellular responses in different organ systems that play important roles in organ development in vivo and stem cell expansion in vitro.

PAPERS IN THIS THESIS

This thesis is based on the following articles and manuscripts, which will be referred to in the text by their Roman numerals (I-IV)

I. Kolterud, Å., Wandzioch, E., and Carlsson, L. (2004) Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2. Gene Expr Patterns, 4, 521-8.

II. Pinto do Ó, P., Kolterud, Å., and Carlsson, L. (1998) Expression of the LIM-homeobox gene LH2 generates immortalized Steel factor-dependent multipotent hematopoietic precursors. EMBO J, 17, 5744-56.

III. Kolterud, Å., Wandzioch, E., Falileeva, L., and Carlsson, L. (2004) Identification and characterization of genes specifically expressed in hematopoietic progenitor/stem cells immortalized by Lhx2. Manuscript

IV. Kolterud, Å., Alenius, M., Carlsson, L., and Bohm, S. (2004) The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity. Development, In press.

The published articles are reproduced with permission from the respective copyright holders.

ABBREVIATIONS

TF transcription factor ECM extra cellular matrix E embryonic day st septum transversum FGF fibroblast growth factor BMP bone morphogenic protein HGF hepatocyte growth factor HS hematopoietic system BM bone marrow HSC hematopoietic stem cell YS yolk sac eryp primitive erythrocyte eryd definitive erythrocyte AGM aorta, gonads and mesonephros bHLH basic helix-loop-helix FACS fluorescence-activated cell sorting GF growth factor SF steel factor Flt3L Flt3 ligand Tpo thrombopoietin ES cell embryonic stem cell LIF leukemia inhibitory factor EB embryoid body CFC colony-forming cell OSN olfactory sensory neuron OE olfactory epithelium OR odorant receptor Ngn1 neurogenin1 OMP olfactory marker protein LIM-HD LIM homeodomain VZ ventricular zone PZ progress zone AER apical ectodermal ridge HPC hematopoietic progenitor cell Dox doxycycline Dkk-1 Dickkopf-1 Mest1 Mesoderm specific transcript1 hMSC human mesenchymal stem cells JNK c-Jun NH2-terminal kinase

8 INTRODUCTION

INTRODUCTION

ORGANOGENESIS

The development of a new individual from a single cell has been a source of wonder throughout history. During the process of development the fertilized egg generates hundreds of different cell types such as liver cells, muscle, blood cells and neurons. The different cell types are organized into complex tissues and organs that are positioned at defined locations in the developing embryo. In this way, the ears always form on the head and the gut in the abdomen and the functionality of the organism is ensured. The initiated tissues and organs also undergo massive growth to generate an organism of correct size and proportion. Although different organs have their specific architecture and cellular composition, the formation of all organs, i.e. the process of organogenesis, has been found to follow a common strategy in which the central theme is cell-cell interactions. All stages of organogenesis, the induction of organ formation, the patterning events ensuring that the cellular components develop in correct spatial and temporal order, the morphogenic movements that shape the organ, and organ expansion depend upon cellular communications. As most organs are formed from cells derived from two distinct tissues, mesenchyme and epithelium, organogenesis thus depends on mesenchymal-epithelial interactions. Studies of the development of a number of different organ systems such as the limbs, lungs and teeth [reviewed in (Martin, 2001; Hogan and Yingling, 1998; Thesleff and Sharpe, 1997)] have revealed that these interactions rely on a combination of intrinsic and extrinsic signals. The initial patterning of an organ is an excellent example of how this is achieved. An initial signal is generally mediated by the mesenchyme via extrinsic signals in the form of secreted or membrane-bound molecules that define the location of neighboring epithelial cells. Through binding to transmembrane receptors on the epithelial cells the extrinsic signals activate intercellular signal transduction pathways that trigger different transcription factors (TFs). Via the activation of TFs, the positional information is interpreted and depending on the intrinsic properties of the responding cell, different cellular responses are triggered. Typical responses are activation of tissue-specific genes, alteration of cell adhesion

9 INTRODUCTION properties or regulation of proliferation. Moreover, the activation of TFs allows the responding cell to respond to the initial signals by producing extrinsic signals itself and thereby establish reciprocal cell-cell interactions. The formation of specific organs thus appears to be based on correct and unique combinations of extrinsic and intrinsic signals.

THE LIVER

The liver is the largest gland in the body and an important component of the digestive system. Situated anterior to the stomach, the liver receives venous blood from the entire gastrointestinal tract and thereby encounters high concentrations of nutrients and toxins absorbed through the intestine. Major functions of the liver are detoxification of metabolic waste products, control of metabolite and serum protein levels in the blood and glycogen storage. In addition to these endocrine activities, the liver has an exocrine function through the production and secretion of bile. Bile is critical for lipid digestion and cholesterol homeostasis, and via the bile, waste products from the metabolic activities of the liver are eliminated. In order to perform these multiple tasks efficiently, the liver has developed a highly specialized architecture [thoroughly described in (Sasse et al., 1992; Kmiec, 2001)]. First, the liver is divided into several lobes covered by a layer of connective tissue called the Glisson’s capsule. Within the lobes, the main functional cells of the liver, the hepatocytes, are organized in anastomosing plates that are usually one or two cells thick (Figure 1). In between the rows of hepatocytes, there are sinusoidal capillaries. These are lined by a fenestrated layer of endothelial cells and separated from the hepatocytes by a narrow space called the space of Disse, through which material is efficiently transferred between the blood and hepatocytes. A number of specialized cell types, collectively called sinusoidal lining cells, line the sinusoidal endothelial cells. Hepatic stellate cells (Ito cells, fat storing cells, lipocytes) are evenly distributed along the sinusoids and extend cytoplasmic processes around and along the endothelial cells of the sinusoids. The stellate cells are involved in transport and storage of vitamin A and are the major source of extracellular matrix (ECM) in the liver. Hepatic macrophages, i.e. Kupffer cells, are also part of the sinusoidal lining cells. As all macrophages,

10 INTRODUCTION

Kupffer cells are phagocytic, and their main function in the liver is to clear gut- derived bacteria, bacterial toxins and damaged erythrocytes. In addition, the Kupffer cells produce a number of mediators that act on both hepatocytes and stellate cells. The liver receives blood from two different sources: nutrient rich blood enters through the hepatic portal vein and oxygenated blood through the hepatic artery. Within the liver, blood from the two sources mixes as the portal vein and hepatic artery both discharge their blood into the sinusoids. After passing through the sinusoidal network, the blood drains to the terminal hepatic vein and enters the circulation. Bile secreted by the hepatocytes is collected in canaliculi that run between adjacent hepatocytes. The canaliculi are small ducts formed by junctional complexes connecting the plasma membranes of opposing hepatocytes. From the canaliculi, the bile flows into bile ducts, which are lined by cholangiocytes. The bile is then transported to the gall bladder via these ducts.

Figure 1. Schematic drawing of liver cell arrangement. Plates of hepatocytes are lined by sinusoids, formed by endothelial cells. In the space between hepatocytes and sinusoids, space of Disse, hepatic stellate cells reside. Kupffer cells are located in the lumen of the sinusoids. Bile canaliculi are located on the lateral surface of adjoining hepatocytes and the bile empty into bile ducts.

11 INTRODUCTION

Early liver development The general architecture of the liver is laid down during embryonic development. Since the structure of the vascular and biliary networks is critical for liver function, it is essential that the cellular components are correctly organized during the course of liver formation. The liver emerges from the ventral foregut endoderm that at the time of liver induction, at around embryonic day (E) 8.5 in mouse, is in close contact with the cardiac mesenchyme and the loose septum transversum (st) mesenchyme (LeDouarin, 1975). Upon interaction with the adjacent mesenchyme, hepatic differentiation and proliferation begin within the gut epithelium, which leads to the formation of a liver bud (LeDouarin, 1975; Cascio and Zaret, 1991; Gualdi et al., 1996;). Around E9.5, the pre-hepatic cells, also called hepatoblasts, delaminate from the gut and migrate as cords into the posterior part of the surrounding st mesenchyme, where the liver will form (LeDouarin, 1975; Fukuda, 1979; Medlock and Haar, 1983). As the hepatoblasts migrate, they closely associate with mesenchymal cells of the st mesenchyme and with endothelial cells lining the liver bud (Cascio and Zaret, 1991; Matsumoto et al., 2001). The endothelial and mesenchymal cells that become captured between the hepatic cords are believed to form the sinusoidal structures and generate the mesenchymal components of the liver, such as the hepatic stellate cells (Enzan et al., 1997). As the architecture of the liver is laid down, the cellular components gradually differentiate into specialized liver cells. The hepatoblasts are believed to be bipotential and give rise both to hepatocytes and cholangiocytes (Blouin et al., 1995; Rogler, 1997; Spagnoli et al., 1998). Liver cell differentiation is accompanied by a massive expansion of the liver. Between E13 and E20 of rat development, the volume of the liver increases 84-fold (Vassy et al., 1988). In parallel with the growth and differentiation activity, the liver is colonized by hematopoietic cells (Houssaint, 1981). From around E10.5 until just before birth, the liver serves as the main hematopoietic organ and provides the microenvironment required for maintenance and expansion of the hematopoietic system (further discussed in the section “The Hematopoietic System”). The liver thus plays an important role in the establishment of two different organ systems.

12 INTRODUCTION

Mesenchymal-epithelial interactions during hepatic induction, morphogenesis and growth As with the development of all visceral organs, formation of the liver is critically dependent upon signaling interactions between the epithelium (i.e. endoderm) and adjacent mesenchyme (Birchmeier and Birchmeier, 1993). The central role of the hepatic mesenchyme during most aspects of liver development has become evident along with the identification of a number of signaling pathways emanating from this tissue. Pioneering tissue culture and transplantation assays indicated that hepatic induction was a two-step process mediated by two distinct mesenchymal tissues, the precardiac mesenchyme and the st mesenchyme (LeDouarin, 1975; Houssaint, 1980; Fukuda-Taira, 1981; Gualdi et al., 1996). The nature of these signals has been identified as the combination of FGF1 and FGF2, produced by the heart mesenchyme, and BMP2 and BMP4, produced by the st mesenchyme (Jung et al., 1999; Rossi et al., 2001) (Figure 2). Although no direct FGF or BMP target genes have been identified in the hepatic endoderm, the homeobox TFs Hex and Prox1 have been shown to be important for the earliest steps of liver development. At the time of hepatic induction, Hex is expressed in a distinct region of the foregut endoderm, which corresponds to the site of the future liver (Keng et al., 1998; Thomas et al., 1998). In the absence of functional Hex, the endoderm is specified to hepatic fate but the liver bud fails to form, indicating that Hex is required for the earliest steps of liver bud formation (Keng et al., 2000; Martinez Barbera, et al.

Figure 2. Overview of early liver development. (A) Liver formation is induced in the ventral foregut endoderm by FGF signaling from the heart mesenchyme in combination with BMP signaling from the st mesenchyme. (B) In response to the inductive signal hepatic proliferation and differentiation is initiated within the endoderm, which leads to the formation of a liver bud. Expression of Hex is required for bud formation. (C) Hepatoblasts migrate away from the gut in a cord-like fashion and intermingle with st mesenchymal cells. Continuous action of BMPs and FGFs is required for the proliferation and outgrowth of the bud. Prox1 is also required for migration, whereas c-met, HGF and Hlx are part of the signals that control liver expansion. [Modified from (Duncan and Watt, 2001)]

13 INTRODUCTION

2000). Prox1 is also expressed in the hepatic endoderm (Oliver et al., 1993) and is required for the migration of the committed hepatoblasts into the st mesenchyme (Sosa-Pineda et al., 2000). The st mesenchyme plays an essential role also after hepatic specification. This has been concluded from st mesenchyme-specific gene inactivation studies resulting in liver expansion and/or morphogenic defects. Owing to these experiments, a number of signaling pathways that regulate liver proliferation and outgrowth have been identified (Figure 2). One of these pathways involves hepatocyte growth factor (HGF), which is secreted from the st mesenchyme (Sonnenberg et al., 1993a; Sonnenberg et al., 1993b). Inactivation of HGF resulted in an embryonic lethal anemia due to liver hypoplasia and apoptosis of the hepatoblasts (Schmidt et al., 1995). A similar phenotype was observed when the HGF receptor c-met, which is expressed by the hepatoblasts, was inactivated (Bladt et al., 1995). These mutant phenotypes indicate that HGF/c-met signaling is required for proliferation and outgrowth of the liver. The divergent homeobox TF Hlx is expressed in the st mesenchyme in a similar pattern to HGF (Lints et al., 1996). Inactivation of Hlx resulted in a more severe liver proliferation defect than in the HGF-/- mice (Hentsch et al., 1996). The absence of apoptosis in Hlx-/- livers suggests that Hlx regulates the expression of some to HGF additional signaling molecule that acts in parallel with HGF. Whether there is some functional connection between Hlx and HGF is yet to be resolved. There are also studies indicating that BMP4 is required for liver bud outgrowth since a delay in the outgrowth of hepatoblasts was observed in BMP4-/- mice (Rossi et al., 2001). The st mesenchyme may also affect the adjacent hepatoblasts via interactions with st-derived ECM. The ECM binds to cells via specific glycoproteins belonging to the integrin cell surface receptor family (Hynes, 1992). The importance of cell-ECM interactions during liver morphogenesis was demonstrated by the inability of β1-integrin-deficient hepatoblasts to migrate and colonize livers in chimeric mice (Fassler and Meyer, 1995). In addition, the liver proliferation defect observed in Smad2+/-; Smad3+/- embryos (Weinstein et al., 2001) was found to be connected to abnormal interactions between hepatoblasts and ECM. ECM molecules may also affect the interactions between st mesenchyme and hepatoblasts by, for example, affecting the availability of secreted signaling molecules.

14 INTRODUCTION

All together, the st mesenchyme and its derivatives appear to play an essential role in multiple aspects of liver development. However, in order to fully decipher the signaling network emanating from this tissue additional factors and pathway components have to be identified.

THE HEMATOPOIETIC SYSTEM

The hematopoietic system (HS) maintains several life-supporting processes such as nutrient and oxygen uptake/transportation and defense against microorganisms. The HS consists of the blood (plasma and blood cells), and the blood forming tissues such as the bone marrow (BM) and thymus. Considering its variable tasks, the HS is made up of a number of specialized cell types that can be categorized into red and white blood cells. The red blood cells, or erythrocytes, that carry oxygen from the lungs to all tissues of the body represent the major cellular component of the blood. The white blood cells, or leukocytes, comprise several different cell types, each with a specific function in the protection against infection. Neutrophils and macrophages are phagocytic cells that protect the body by ingesting foreign particles and bacteria. Macrophages also scavenge dead cells and tissue debris. B- lymphocytes are specialized in antibody production whereas T-lymphocytes are involved in cell-mediated immunity. Megakaryocytes produce platelets that are important in the process of blood clotting. During adult life, the BM serves as the major hematopoietic organ and all blood cells ultimately have their origin in the BM, although maturation of T-lymphocytes takes place in the thymus. All mature blood cells have a limited life span, ranging between less than a day for neutrophils and around 43 days for erythrocytes (in mice) (Moore, 1997). Maintenance of the HS thus requires constant renewal of hematopoietic cells. This continuous production of blood cells is maintained by a small population of hematopoietic stem cells (HSCs) that reside in the bone marrow. The HSCs generate immature progenitors that progress through a number of intermediate stages with gradually more restricted differentiation and proliferation potential until they differentiate into functional blood cells. A schematic view of the hematopoietic hierarchy is presented in Figure 3.

15 INTRODUCTION

Figure 3. Schematic representation of the hematopoietic hierarchy. The hematopoietic stem cell is in the foundation of the hierarchy. The HSC can self-renew (indicated by the arrow above the stem cell) and give rise to all differentiated lineages of blood cells. Between the HSC and the mature blood cells are progenitors with progressively restricted potential for differentiation and proliferation. Adapted from (Keller, 1992).

16 INTRODUCTION

Development of the hematopoietic system During embryogenesis the HS develops in successive steps in distinct organs. The first wave of hematopoiesis occurs in the extra-embryonic yolk sac (YS) at around E7.5 (Metcalf and Moore, 1971; Russell, 1979). The YS hematopoiesis is transient, and at E11-12 hematopoiesis shifts to the fetal liver. The fetal liver serves as the main hematopoietic organ until around birth when the BM becomes the prevalent site of hematopoiesis (Russell, 1979; Ema and Nakauchi, 2000). During the course of development and depending on the anatomical site and developmental stage, the properties of the HSCs vary. The first wave of hematopoiesis is initiated by early HSCs (Wong et al., 1986), that produce a transient burst of large nucleated erythrocytes known as primitive erythrocytes (eryp), as they express an embryonic type of hemoglobin (Bauer et al., 1975; Brotherton et al., 1979). Around the time that hematopoiesis is shifted to the liver, definitive enucleated erythrocytes (eryd) expressing the adult type of hemoglobin (Barker, 1968; Brotherton et al., 1979) emerge and gradually become the predominant form of erythrocytes. The origin of the definitive HSCs that give rise to the definitive (adult type) of hematopoiesis has been the subject of controversy [reviewed in (Kyba and Daley, 2003)]. Recent work has, however, suggested that definitive HSCs, seeding the fetal liver, originates both from the YS and the region that later gives rise to the aorta, gonads and mesonephros (AGM) (Yoder et al., 1997; Matsuoka et al., 2001; Palis et al., 2001; Kumaravelu et al., 2002). The genetic regulation of hematopoietic development is not very well understood. A number of players in the molecular cascade that specifies the hematopoietic lineage have though been identified, mainly through gene inactivation studies. The zink finger TF GATA-2 and the basic helix-loop-helix (bHLH) TF Tal-1/SCL have been shown to be essential for the earliest steps of HS development. SCL is required for both primitive and definitive hematopoiesis, which makes it a hematopoietic master regulator (Robb et al., 1995; Porcher et al., 1996). The activity of SCL depends on physical interaction with its binding partner LMO2, and disruption of LMO2 results in a defect similar to that in the SCL mutant (Warren 1994, Yamada 1998). GATA-2 is also required for YS hematopoiesis (Tsai et al., 1994). Other TFs such as the core binding heterodimeric TF AML1:CBFβ and c-myb act at a later stage in hematopoietic development and are specifically required for the development and/or expansion of definitive

17 INTRODUCTION hematopoiesis (Mucenski et al., 1991; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996).

Hematopoietic stem cell phenotype The capacity of the HSCs to continuously supply the HS with mature blood cells without being depleted depends on their ability to generate new stem cells in a process referred to as self-renewal. The HSCs are thus defined as cells with the potential to both self-renew and differentiate into all types of mature hematopoietic cells (Jordan and Lemischka, 1990; Smith et al., 1991; Osawa et al., 1996). This unique capacity has been demonstrated by the ability of a single HSC to regenerate and maintain the entire HS of a lethally irradiated host (Morrison et al., 1995; Osawa et al., 1996; Krause et al., 2001). Within the hematopoietic hierarchy the HSCs constitute a rare population with a frequency of only one in 1 x 105 BM cells (Trevisan et al., 1996; Boggs, 1999). Despite this low frequency and the lack of unique markers, methods have been developed that allow for enrichment of HSCs from total BM. These methods are based on cell surface staining with a combination of monoclonal antibodies and fluorescence-activated cell sorting (FACS) [reviewed in (Wognum et al., 2003)]. The HSCs are distinguished from mature hematopoietic cells by their lack of lineage-specific markers (lin) such as Ter119 (erythrocytes), Mac-1/Gr-1 (myeloid cells), CD4 (T lymphocytes), and B220 (B lymphocytes) and their expression of a set of stem cell markers. In the mouse, HSCs have been shown to express the cell-surface markers c-kit, Sca-1 and Thy-1 (Spangrude et al., 1988; Uchida and Weissman, 1992; Osawa et al., 1996). By selecting for cells that express a combination of these markers and lack expression of lineage markers, i.e. lin-, Sca-1+, Thy-1lo, c-kit+, an almost pure population of BM HSCs can be isolated. HSCs isolated from fetal liver and from YS differ in their expression of cell-surface markers as compared to adult HSCs. YS HSCs have been shown to express CD34, c-kit, Mac-1 and AA4.1, and to be negative for Sca-1 (Yoder et al., 1997). In contrast, fetal liver HSCs express c-kit, Mac-1, AA4.1 and Sca-1 (Morrison et al., 1995; Sanchez et al., 1996). As Mac-1 and AA4.1 are not detected on adult HSCs, these can be considered to be markers for embryonic HSCs.

18 INTRODUCTION

The role of the microenvironment in HSC self-renewal Although the ability to self-renew is the most fundamental property of the HSCs, most aspects of self-renewal are unsolved. This can probably be explained by the fact that stem cells in the BM, in addition to being scarce, are also largely quiescent (Bradford et al., 1997; Cheshier et al., 1999). Although the molecular mechanisms that regulate HSC dynamics remain elusive, it is clear that HSC self-renewal to a large extent depends on extrinsic signals in the form of cell-cell interactions, cell- ECM interactions and soluble growth factors (GFs). To date, all attempts to recapitulate HSC self-renewal in culture for a prolonged time have been unsuccessful. This indicates that their ability to self- renew depends on elements specifically present in their normal environment. Within the hematopoietic organs, the HSCs are in close contact with a heterogeneous population of support cells, referred to as stromal cells. The stromal cells create a complex microenvironment that provides both structural and molecular support, and influences hematopoietic stem/progenitor cells via cell-cell interactions, expression of adhesion molecules, secretion of GFs and production of ECM (Tavassoli, 1991; Rafii et al., 1997; Torok-Storb et al., 1999). The importance of a normal hematopoietic microenvironment was nicely demonstrated in the Sl/Sld mouse (Galli et al., 1994). Stromal cells from these mice are incapable of producing the membrane-bound form of Steel factor (SF) (Flanagan and Leder, 1990; Fleischman et al., 1995), the ligand for the tyrosine-kinase receptor c-kit. This deficiency resulted in an anemia that could not be rescued by transplantation of wild-type hematopoietic cells (Russell et al., 1959). The hematopoietic defect was thus strictly due to a deficient environment. In addition to SF, a number of membrane-bound and soluble stromal factors with an effect on hematopoietic progenitor/stem cells in vitro, and thus likely to play important role in regulation of HSCs in vivo, have been identified. It has, for example, been demonstrated that a combination of the Flt3-ligand (Flt3L), SF, IL-6 and thrombopoietin (Tpo) has the ability to enhance proliferation of early progenitors in vitro (Sui et al., 1995; Piacibello et al., 1997; Yagi et al., 1999; Kimura et al., 2000; Lazzari et al., 2001; Rappold et al., 1999; Ueda et al., 2000) although their capacity to promote HSC self-renewal is limited. The only considerable expansion of HSCs under physiological conditions occurs in the fetal liver during embryonic development. A 38-fold increase in the

19 INTRODUCTION number of HSCs has been observed in the liver between E12-E16 (Ema and Nakauchi, 2000). Moreover, isolated fetal liver HSCs have been found to have a greater proliferative capacity as compared to adult BM stem cells (Rebel et al., 1996). This suggests that stromal cells from different organs may have different capacities for supporting self-renewal and that the extrinsic signals that promote extensive expansion of HSCs are likely to be produced by the cellular components of the fetal liver microenvironment. The understanding of the molecular mechanisms that regulates expansion of HSCs in the fetal liver would thus give insights into how HSC self-renewal is regulated.

ES cells differentiated in vitro mimic early hematopoietic development Studies of early hematopoietic development have been hampered by the inaccessibility and limited number of hematopoietic cells in early embryos. The establishment of a model system based on embryonic stem (ES) cells differentiated in vitro has provided a way of overcoming these difficulties. ES cells are pluripotent and isolated from the inner cell mass of mouse blastocysts (Evans and Kaufman, 1981; Martin, 1981). When maintained in the presence of leukemia inhibitory factor (LIF), ES cells have an unlimited capacity for self-renewal and can be cultured without losing their pluripotent capacity. This has been demonstrated by their ability to contribute to all tissues, including the germ line, upon reintroduction into host blastocysts (Bradley et al., 1984). In addition, ES cells have the ability to differentiate in vitro [reviewed in (Keller, 1995)]. Upon removal of LIF, ES cells spontaneously differentiate and aggregate to form so- called embryoid bodies (EBs). The EBs generate cells representing all three germ layers and develop precursors for a number of different cell lineages such as hematopoietic, neuronal, endothelial and muscle. Hematopoietic development within the EBs is both efficient and reproducible, and not dependent on addition of any GFs other than those present in fetal calf serum (Doetschman et al., 1985). The development of hematopoietic progenitors within the EBs is generally examined in clonal assays (Metcalf and Nicola, 1984) in which dissociated EBs are plated on semisolid culture medium. This provides physical separation of plated cells and ensures single cell origin for the colonies that form. The progenitors that have the ability respond to the combination of hematopoietic GFs chosen for a particular

20 INTRODUCTION assay will proliferate, differentiate and form colonies, and are therefore referred to as colony-forming cells (CFC). Depending on the type of mature cells that the colony contains, the progenitors are classified retrospectively and named after their colony content. The immediate precursors of mature hematopoietic cells generate single-lineage colonies; bipotent progenitors generate colonies containing mature cells of two different lineages, whereas multipotent progenitors generate colonies containing several different cell lineages. Similar to hematopoietic development within the embryo proper, eryp are the first hematopoietic progenitors to develop within the EBs, followed by eryd and definitive myeloid progenitors (Keller et al., 1993). The EB thus recapitulates the events involved in establishment of the earliest hematopoietic cells in vivo. So far, all attempts to reproducibly generate HSCs with repopulating potential from genetically unaltered ES cells have failed. This could be due to the fact that the early HSCs are unable to function efficiently in an adult environment. Based on the demonstration that YS-derived HSCs that normally fail to repopulate adult mice could be transplanted into and functionally repopulate newborn mice (Yoder and Hiatt, 1997) it is believed that YS HSCs may lack the expression of surface molecules required for homing to the BM. Since ES differentiation most closely reflects YS hematopoiesis, any eventually ES-derived HSCs may in a similar way lack the properties required for homing to the BM.

THE OLFACTORY SYSTEM

The nose has the ability to detect and discriminate among thousands of odors, allowing the identification of both pleasures such as food and drinks and dangers such as smoke and spoiled food. The odors are recognized by olfactory sensory neurons (OSNs) positioned in the olfactory epithelium (OE) that line the walls of the nasal cavity [reviewed in (Axel, 1995)] (Figure 4A). As odorous molecules enter the nose, they dissolve in the layer of mucus that covers the epithelium and bind to specific odorant receptors (ORs) expressed by OSNs. Upon receptor binding, a signal transduction process is initiated which generates an action potential that is conducted along the olfactory nerve to the olfactory bulb in the brain. Projection neurons in the bulb then send olfactory information to higher

21 INTRODUCTION subcortical and cortical regions in the brain where the information is further processed and encoded.

Organization and development of the olfactory epithelium The olfactory epithelium is organized into a layered structure in which the neurons are arranged according to increasing maturity as one proceeds from basal to apical cell layers (Figure 4). As a result of this organization, the neuronal progenitor containing population of globose basal cells (Caggiano et al., 1994) is together with the horizontal basal cells located in the basal layer of the OE, adjacent to the basal lamina that separates the epithelium from the lamina propria. Immature neurons are positioned just above the basal cells, while more mature bipolar OSNs, which have extended their axons to the olfactory bulb, have migrated up towards the surface where they constitute several layers. The most apical part of the epithelium, lining

A B

Figure 4. Organization of the OE. (A) In situ hybridization of a coronal section through the nasal cavities of an adult mouse visualizing the olfactory epithelium. (B) Cartoon of a section through the OE of an adult mouse illustrating the positions of the different cell types. Sustentacular cells (SUS) are located closest to the nasal cavity. Underneath the SUS cell layer, mature OSNs, that protrude ciliated dendrites to the surface of the OE and axons basally towards the olfactory bulb reside, while immature OSNs are located more basally. Globose basal cells (GBC) and horizontal basal cells (HzBC) are located in the most basal part of the OE, adjacent to the basal lamina.

22 INTRODUCTION the nasal cavity, contains a layer of supporting cells. These cells, called sustentacular cells, are not neurons but cell lineage analysis suggests that sustentacular cells and neurons originate from a common progenitor (Goldstein et al., 1998; Huard et al., 1998; Chen et al.,2004). The OE develops from distinct ectodermal thickenings, so-called olfactory placodes that appear around E9.5 on the ventrolateral aspects of the head. After placode induction, which is believed to be mediated by morphogenic signals emanating from adjacent tissues (Webb and Noden, 1993), the placodes invaginate to form the olfactory pits. It is from the ectoderm of the invagination that the OE is formed and olfactory sensory neurons are subsequently generated (Cuschieri and Bannister, 1975). Most of the important features of the OE are established by E13, and by then the layered organization can be readily distinguished. At early developmental stages, the epithelium mainly consists of immature neurons but as organogenesis proceeds, the number of mature OSNs and the thickness of the OE increases so that it reaches its normal size after birth (Cuschieri and Bannister, 1975).

Development and differentiation of OSNs Compared to most regions of the nervous system, the OE is unique in that it is regenerated throughout life (Graziadei and Graziadei, 1979a; Graziadei and Graziadei, 1979b). Similar to the keratinocytes in epidermis, dying or damaged OSNs are constantly replaced by newly generated neurons. Olfactory neurogenesis is thus an ongoing process which can be studied in the adult OE as well as during embryonic development. The regenerative capacity of the OE is maintained by a population of oligopotent progenitor cells that reside within the globose basal cell layer (Goldstein et al., 1998; Huard et al., 1998; Caggiano et al., 1994; Chen et al., 2004). The progenitors generate neuronal daughter cells that mature to OSNs as they migrate apically within the OE. Although the precise molecular events controlling OSN differentiation are unclear, the basic helix-loop-helix (bHLH) transcriptional regulators Mash1, Neurogenein1 (Ngn1) and NeuroD have been shown to be part of the genetic control of olfactory neurogenesis. These TFs are expressed in dividing progenitor cells from the earliest stages of OE development (Cau et al., 1997) (Figure 5). Mash1 is required at an early stage in the OSN lineage for the generation of a subset of dividing progenitors in the basal layer

23 INTRODUCTION

(Guillemot et al., 1993; Cau et al., 2002). Ngn1 acts downstream of Mash1 and has been found to be essential for the activation of a differentiation program in the basal progenitors (Cau et al., 2002). The action of Ngn1 is presumably exerted by activation of the differentiation gene NeuroD, which is transiently expressed during the transition between proliferation and onset of differentiation (Nibu et al., 1999). As the neuronal progenitors leave the cell cycle, they begin to express pan- neuronal markers such as SCG10, β-Tubulin III, GAP43 and NCAM (Stein et al., 1988; Geisert and Frankfurter, 1989; Verhaagen et al., 1989; Mahanthappa and Schwarting, 1993) and to project their axons towards the olfactory bulb. The gradual transformation of the neurons from an immature to a mature stage is marked by the expression of the olfactory marker protein (OMP) (Danciger et al., 1989). How the maturation process is regulated remains to be determined. However, the onset of OR expression has been shown to occur prior to the transition to mature OSNs (Iwema and Schwob, 2003) and is believed to be a prerequisite for the neurons to complete their differentiation. OR expression can be detected from E11.5-12.5 during embryonic development (Sullivan et al., 1995).

Figure 5. Differentiation of olfactory sensory neurons. Oligopotent progenitors are believed to generate both neurons and sustentacular cells. Mash1 and Ngn1 are expressed in immediate neuronal progenitors. NeuroD is transiently expressed as the neurons exit the cell cycle. Immature OSNs express pan neuronal markers such as SCG10, β-Tubulin III, GAP43 and NCAM and begin to project their axons towards the bulb. Onset of OR gene expression occurs prior to expression of OMP, which is a marker for mature OSNs.

24 INTRODUCTION

Odorant receptors The initial step of odor recognition and discrimination is mediated by odorant receptors expressed by OSNs. Isolation of the OR genes revealed that the ORs are G-protein coupled receptors encoded by a large multigene family (Buck and Axel, 1991). Screening of completely sequenced genomes has suggested that the mouse genome encodes 1000-1300 different odorant receptors, which correspond to about 1% of the genome (Young et al., 2002; Zhang and Firestein, 2002). This makes the OR family the largest gene family identified so far, and thus emphasizes the significance of the olfactory system in mammals. Examination of OR gene expression by in situ hybridization resulted in a number of intriguing discoveries. First of all, each OR gene is expressed in approximately 0.1% of the OSN population (Ressler et al., 1993; Vassar et al., 1993). This suggests that each neuron only expresses one or a small number of ORs [reviewed in (Mombaerts, 2004)], a hypothesis supported by results from single-cell rt-PCR analysis (Malnic et al., 1999). Moreover, the absence of OSN clusters expressing the same OR gene indicates that OR gene selection is not intrinsic to the OSN progenitors, but rather an event occurring at a relatively late differentiation stage (Vassar et al., 1993). In addition, the expression of OR genes has been found to be driven exclusively from one of the two alleles (Chess et al., 1994). The regulation of monoallelic expression was recently found to involve the receptor proteins themselves (Serizawa et al., 2003; Lewcock and Reed, 2004). According to the suggested model, each OSN stochastically activates one OR gene promoter and the OR protein produced prevents expression from other OR loci via a feedback signal. How the OR protein- mediated negative control is achieved is, however, still not understood. It has been suggested though that the ORs may function in a manner similar to that of the pre- B cell receptor in the immune system. During B cell development the pre-B cell receptor ensures that each B cell expresses only one B cell receptor through a mechanism referred to as allelic exclusion (Schweighoffer et al., 2003).

OE zones Examination of OR gene expression further revealed that the OE is divided into a number of spatially restricted zones (Ressler et al., 1993; Vassar et al., 1993; Sullivan et al., 1995). With a few exceptions, every OR gene is restricted in expression to one of these zones. Within the zones neurons expressing the same

25 INTRODUCTION

OR are broadly distributed and scattered so that they intermingle with OSNs expressing other ORs belonging to the same zonal set. The zones are organized from the dorsomedial to the ventrolateral part of the nasal cavity (Figure 6) and zonal segregation is similarly to the layered organization (discussed earlier) apparent as early as at E13 (Sullivan et al., 1995). The mechanisms controlling zonal specificity are still largely unknown. However, based on how cell specification is regulated in other systems (Edlund and Jessell, 1999), it is likely that zonal specification of the OE is determined by a combination of intrinsic and extrinsic signals. Depending upon positional information generated from the surroundings and/or intrinsically determinants within the basal progenitors, newly borne OSNs may be restricted to expressing ORs from a certain zone-specific set. A role for the local environment in selection of OR gene expression has been supported by the discovery that zonal organization applies to most cells in the OE – including the basal cells, sustentacular cells, and the underlying mesenchyme (Miyawaki et al., 1996; Norlin et al., 2001; Oka et al., 2003; Gussing and Bohm, 2004; Whitby-Logan et al., 2004). To date, the functional significance of the spatial restriction in OR gene expression is not fully understood. A similar organization of the bulb together with the observation that neurons belonging to a given zone converge their axons to a corresponding zone in the bulb suggest that the zonal segregation may be a way of organizing information between the OE and bulb (Sullivan and Dryer, 1996).

Figure 6. Schematic illustration of the main OR expression zones. In situ hybridization with an Lhx2 RNA probe on a coronal OE section of an E15 mouse. Lhx2 is expressed throughout the epithelium. The zonal organization is illustrated by the dotted line. DV, dorsomedial zone, VL, ventrolateral zone. The VL can be further divided in to 3 zones, then referred to as zone 2-4

26 INTRODUCTION

Since the selective expression of an OR gene is central to the function of an OSN, a number of attempts have been made to reveal the mechanisms controlling zonal expression of OR genes, in particular by searching for regulatory regions and TF-binding sites upstream of OR genes. Various 5’ sequences have been isolated and tested for their ability to recapitulate correct expression of a specific OR gene in transgenic mice [reviewed in (Kratz et al., 2002)]. The length of the upstream sequences required for driving normal OR expression has varied from around 100 kb to as little as 400 base pairs (Qasba and Reed, 1998; Serizawa et al., 2000; Vassalli et al., 2002; Hoppe et al., 2003). This perhaps indicates that OR expression is regulated both via some locus control motif at a distance from the OR genes, and through regulatory motifs located immediately upstream of the OR genes, a hypothesis that remains to be proven experimentally. Although a number of putative TF binding sites have been detected in the upstream region of OR genes, the relevance of these in regulation of OR expression is still unclear (Sosinsky et al., 2000; Hoppe et al., 2003). The mechanism that regulates the choice of OR gene expression from a zonally restricted set of OR genes thus remains elusive.

LIM-HD TFs AND REGULATION OF PATTERNING AND CELL SPECIFICATION

One family of transcription factors that plays a critical role during several different aspects of embryonic development is the LIM homeodomain (LIM-HD) TF family [reviewed in (Curtiss and Heilig, 1998; Hobert and Westphal, 2000)]. The characteristic features of these TFs are two highly conserved LIM domains at the N-terminal end followed by a DNA-binding homeodomain. The LIM domain is a cysteine-rich zinc finger-like motif that mediates protein-protein interactions and was named by the initials of the three founders of the gene family, Lin-11, Islet-1 and Mec-3 (Freyd et al., 1990; Karlsson et al., 1990; Way and Chalfie, 1988). Loss-of-function studies have demonstrated that LIM-HD TFs are involved in a wide variety of developmental processes, such as early embryonic patterning, cell and tissue specification and neuronal differentiation [reviewed in (Curtiss and Heilig, 1998; Hobert and Westphal, 2000)]. Examples of LIM-HD TF family members involved in the establishment of the pattern of an organism or tissue are

27 INTRODUCTION

Lim1/Lhx1 and Lmx1. During vertebrate development, the organizer region (Spemann organizer in Xenopus) induces and patterns the body axis (Schweitzer and Tabin, 1999). Lim-1/Lhx1 is expressed in the organizer (Taira et al., 1994; Shawlot and Behringer, 1995) and is an essential regulator of the head organizer activity since targeted deletion of the Lim-1 gene resulted in a lack of head structures (Shawlot and Behringer, 1995). Lmx1 is expressed in the dorsal mesenchyme of the limb bud and is required for the specification of dorsal patterning in the developing limb (Riddle et al., 1995; Vogel et al., 1995). Several LIM-HD proteins are involved in cell fate decisions by regulating tissue-specific genes. Lim3/Lhx3 is for example expressed in the developing pituitary gland (Zhadanov et al., 1995) where it controls a critical step of pituitary fate commitment by activating transcription of several pituitary-specific promoters (Bach et al., 1995; Sheng et al., 1996). Most members of the LIM-HD family are also expressed in the developing nervous system. Being expressed in discrete subsets of developing neurons, LIM-HD TFs are involved in specification of neuronal identity (Tsuchida et al., 1994; Lumsden, 1995; Thor et al., 1999). Evidence for a specific LIM code in the determination of neuronal cell fate comes from studies of motor neurons in the spinal cord. Different motor neuron subtypes are specified by the combinatorial expression of LIM-HD TFs. A set of motor neurons that will project their axons ventrally is for example defined by the transient expression of Lhx3 and Lhx4 in combination with Isl1 (Sharma et al., 1998; Thor et al., 1999). In addition to different LIM-HD family members functioning at different developmental stages, there are examples of individual LIM-HD genes that act at multiple stages of embryonic development. The Drosophila gene apterous (ap) is, for instance, involved in both organ patterning and cell fate determination. During Drosophila wing development, ap expression is restricted to the dorsal compartment of the wing disc where it controls dorsal-ventral polarity as well as wing outgrowth (Cohen et al., 1992; Diaz-Benjumea and Cohen, 1993; Blair et al., 1994). In addition, apterous is required for interneuron pathway selection and specification of muscle precursors (Bourgouin et al., 1992; Lundgren et al., 1995) The ability of a single LIM-HD TF to regulate distinct target genes in the development of different tissues is not fully understood. However, the identification of the LIM domain as a protein-protein interaction domain together

28 INTRODUCTION with the isolation of a number of LIM domain interacting factors have suggested that the activity of LIM-HD TFs is regulated by recruitment of additional proteins into multiprotein complexes (Curtiss and Heilig, 1998; Bach, 2000). This way, depending on the available binding partners, the same LIM-HD protein could regulate different developmental events at different time points and tissues.

Lhx2 AND ORGANOGENESIS

A LIM-HD TF of particular interest, due to its pivotal role in a wide variety of developmental events, is the apterous ortholog Lhx2 (Xu et al., 1993; Porter et al., 1997). Like most members of the LIM-HD protein family, Lhx2 participates in the development of the nervous system. During neurogenesis, Lhx2 expression is detected throughout the telencephalic cortex in the developing forebrain and in the outer layers of the dorsal midbrain and rostral hindbrain. Lhx2 is also expressed in the spinal cord, olfactory epithelium and in the retina of the developing eye (Xu et al., 1993; Porter et al., 1997). In the absence of functional Lhx2, there is a drastic reduction in size of the cerebral cortex and eye development is arrested prior to the formation of the optic cup (Porter et al., 1997). BrdU studies of Lhx2 mutant brains have revealed a decreased proliferation of cortical ventricular zone (VZ) progenitors in the hippocampus and neocortex (Porter et al., 1997). In addition, at the expense of cortical neuroepithelium, a drastic expansion of the choroid plexus and the cortical hem – which is believed to serve as a specialized organizing center for hippocampal development – was observed (Bulchand et al., 2001; Monuki E.S. et al., 2001). It thus appears as if Lhx2 is involved in regulating proliferation of neuronal progenitors and is required for the patterning of the dorsal telencephalon and cerebral cortex. During patterning of the different neuronal subtypes in the spinal cord, Lhx2 is expressed in a subpopulation of postmitotic dorsal sensory interneurons (Helms and Johnson, 1998; Lee et al., 1998) and may have a role the specification of that particular set of neurons. However, the direct involvement of Lhx2 in interneuron differentiation has not been determined and no interneuron specification defect in the Lhx2-/- mouse has been reported. In addition to reduced forebrain and lack of eyes, the Lhx2-/- mice displayed another major phenotypic abnormality. The livers were 7-fold smaller and pale as

29 INTRODUCTION compared to wild-type mice, although the overall histology appeared normal (Porter et al., 1997). The impaired liver development of the Lhx2 null mice affected the ability of the liver to support hematopoiesis and lead to a defective definitive hematopoiesis. This defect caused anemia which resulted in embryonic death at around E16. That the hematopoietic defect was due to a deficient microenvironment and not intrinsic to the hematopoietic cells was confirmed by transplantation assays where Lhx2-/- HSCs were able to generate cells of all hematopoietic lineages in wild-type recipients (Porter et al., 1997). The effect of Lhx2 on hematopoiesis is thus cell non-autonomous. The cloning of Lhx2 from a pre-B cell library suggested that the scattered expression of Lhx2 observed in the fetal liver was in developing lymphoid cells (Xu et al., 1993). However, the requirement of Lhx2 for normal fetal liver development implies that Lhx2 is expressed in non-hematopoietic liver cells. Therefore, the exact location and timing of Lhx2 expression within the liver microenvironment remains to be investigated. Lhx2 is also expressed in the mesenchymal progress zone (PZ) of the limb bud (Rodriguez-Esteban et al., 1998; Rincon-Limas et al., 1999). Studies in chick showed that mis- expression of Lhx2 in the limb bud leads to ectopic expression of genes involved in formation of the apical ectodermal ridge (AER), a major signaling center during limb development. Furthermore, expression of a dominant negative Lhx2 led to defective AER formation and arrested limb outgrowth (Rodriguez-Esteban et al., 1998). Lhx2 thus appears to regulate limb outgrowth in a similar way as apterous regulates wing outgrowth. The Lhx2-/- mice do not display any limb phenotype though (Porter et al., 1997), presumably due to functional overlap since other closely related LIM-HD proteins, like Lhx9 and Lmx1b, show overlapping expression pattern in limb mesenchyme (Riddle et al., 1995; Vogel et al., 1995; Bertuzzi et al., 1999), and the dominant negative mutation of Lhx2 in the chick system might interfere with the function of all these LIM-HD proteins. Taken together, Lhx2 plays a central role in a variety of developmental processes, which emphasizes a similar significance of Lhx2 in mammalian development as apterous in Drosophila development. Analyses of the Lhx2 function during embryonic development might therefore shed light on basic molecular mechanism regulating organ development.

30 AIMS

AIMS

The aim of this thesis was to analyze different aspects of embryonic development such as regulation of stem cells, organ expansion/organization and tissue specification. More specifically:

• To determine in what cells Lhx2 is expressed during liver development and to analyze the role of Lhx2 in this process by comparing early liver development in Lhx2-/- and wild-type embryos

• To analyze the role of Lhx2 during the formation and expansion of the hematopoietic system by expressing Lhx2 in ES cells differentiated in vitro

• To analyze the function of Lhx2 in the formation of the olfactory epithelium during embryonic development by comparing Lhx2-/- and wild- type embryos

• To isolate genes putatively linked to Lhx2 function

31 RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

The LIM-HD transcription factor Lhx2 has been shown to trigger a variety of cellular responses during the development of different organ systems, via both cell autonomous and cell non-autonomous mechanisms. This wide-ranging activity of Lhx2 suggested that elucidation of Lhx2 function could be a way of improving our knowledge of how transcriptional regulation and cell-cell signaling interact to control organogenesis. In this thesis, I have analyzed the role of Lhx2 during the development of three different organ systems, the liver, hematopoietic system and the olfactory epithelium. My aim was to gain insights into how these organs are formed by studying the function of Lhx2 in the respective organ systems.

Lhx2 AND LIVER DEVELOPMENT (paper I)

Liver development is based on interactions between the foregut endoderm and adjacent mesenchymal tissues. The septum transversum mesenchyme plays an essential role in this process by producing soluble signaling molecules that induce differentiation, proliferation and migration of the neighboring gut endoderm (Schmidt et al., 1995; Rossi et al., 2001; Weinstein et al., 2001). Although a number of extrinsic signals involved in the mesenchymal-epithelial interactions have been identified, very little is known about the intrinsic signals required for these interactions. The reported expression of Lhx2 by uncharacterized fetal liver cells and the liver expansion defect observed in Lhx2-/- mice (Xu et al., 1993; Porter et al., 1997) suggested that Lhx2 is important for proper liver development and perhaps contribute to the intracellular conditions required for the cell-cell interactions that control liver development.

Lhx2 is expressed in hepatic mesenchyme To understand the role of Lhx2 during liver development, we performed a detailed expression analysis of Lhx2 from the time of hepatic induction until a few weeks after birth using in situ hybridization. No Lhx2+ cells were detected in the ventral foregut region prior to, or at liver induction. However, at E9, when the liver bud

32 RESULTS AND DISCUSSION had formed, expression of Lhx2 was observed in the st mesenchyme that surrounds the liver bud. This suggests that signals from the liver bud induce Lhx2 expression in the adjacent st mesenchyme. Moreover, the distinct expression of Lhx2 in the st mesenchyme enclosing the liver bud defines Lhx2 as the first TF specifically expressed in hepatic mesenchyme, and suggests that Lhx2 might be a useful marker for this tissue. As the liver bud proliferated, cords of hepatoblasts migrated out into and intermingled with the Lhx2+ mesenchymal cells. As a result, Lhx2- expressing mesenchymal cells became integrated into the developing liver and were later found scattered within the liver and in a discontinuous layer surrounding each liver lobe. The scattered expression of Lhx2 within the liver lobes was maintained in the adult liver, whereas expression in cells lining the liver was less apparent from E15. The nature of the Lhx2+ cells in the adult liver was determined by immunohistochemical analysis using different mesenchymal cell markers. Co- expression of Lhx2 and desmin, a marker for hepatic stellate cells (Yokoi et al., 1984; Tsutsumi et al., 1987), revealed that Lhx2 in the adult liver is expressed in a population of hepatic stellate cells. Taken together, these data demonstrate that Lhx2 is specifically expressed in the liver-associated st mesenchyme and that these cells become integrated in the developing liver where they contribute to a subpopulation of hepatic stellate cells in adult liver. Our findings provide evidence that at least part of the hepatic stellate cell population originates from the st mesenchyme and thus bring some clarity into the debated origin (mesenchymal versus neural crest) of the hepatic stellate cells (Sato et al., 2003). The lineage relationship between Lhx2+ and Lhx2- stellate cells, if any, remains to be elucidated.

The mutant phenotype is due to defective mesenchyme Gene inactivation studies have identified a number of regulatory factors and signaling molecules with important functions in liver organogenesis (Bladt et al., 1995; Schmidt et al., 1995; Hentsch et al., 1996; Keng et al., 2000; Sosa-Pineda et al., 2000) (discussed earlier). Embryos with defective liver development usually die in utero around E13.5-15.5, due to anemia as the defective livers fail to support hematopoiesis. As inactivation of Lhx2 gives a similar embryonic lethal phenotype (Porter et al., 1997) we wanted to exclude that the liver expansion defect in Lhx2-/- mice was due to lack of expression of any of the so far identified regulators of liver

33 RESULTS AND DISCUSSION development. We therefore analyzed Lhx2-/- livers for the expression of genes with known functions in liver organogenesis, such as Hlx, HGF, BMP2, Hex, Prox1, and c-met (Figure 7A). The lack of obvious differences in expression as compared to wild-type expression indicated that Lhx2 acts via some additional and parallel pathway which may involve novel intrinsic and/or extrinsic factors. The absence of Lhx2 expression in the foregut region at the time of liver specification confirmed that Lhx2 is not involved in liver induction. However, the early onset of Lhx2 expression in the st mesenchyme and the close interaction between the Lhx2+ mesenchyme and the liver bud (Figure 7B) suggested a role for Lhx2 in the mesenchymal-epithelial interactions that govern early stages of liver development. In support of this hypothesis, we found that the liver expansion defect previously described (Porter et al., 1997) was apparent as early as E10.5. Moreover, cells that normally express Lhx2 were present in the mutant livers, which indicated that Lhx2 was not required for the generation of this cell population. Hence, the liver expansion phenotype is not due to the lack of a cell population. Together, these results indicate that Lhx2 is required in the hepatic mesenchyme at early developmental stages for normal liver expansion and that the mutant phenotype is due to the presence of a defective mesenchymal cell population, most likely consisting of altered hepatic stellate cells. This suggests, therefore, that Lhx2 regulates expansion of the hepatic epithelium by a cell non- autonomous mechanism. Such a role for Lhx2 is in accordance with the described function of Lhx2 in chick limb development (Rodriguez-Esteban et al., 1998). During limb development, Lhx2 is expressed in the limb progress zone and involved in the reciprocal mesenchymal-epithelial signaling required for limb outgrowth. The Lhx2-dependent induction and maintenance of the epithelial AER is believed to be achieved via diffusible factor(s).

Possible mechanisms of Lhx2-mediated liver expansion Although it is clear from our observations that expression of Lhx2 in the hepatic mesenchyme is required for proper expansion of the liver, the exact role of Lhx2 in this process remains unclear. It is possible that Lhx2 functions by directly or indirectly regulating some extrinsic signal that induces proliferation in the neighboring hepatic cells (Figure 7C). In other words, Lhx2 may regulate a soluble mediator by binding to its promoter and activating transcription (directly), or by

34 RESULTS AND DISCUSSION activating/repressing the transcription of some other gene that in turn leads to the synthesis or activation of a soluble molecule (indirectly). Upon secretion from the Lhx2-expressing mesenchymal cells and by binding to receptors on neighboring epithelial cells, the soluble mediator could affect the proliferation of these cells. Lhx2 would thus act via a similar type of soluble mediator as that suggested in chick limb development (Rodriguez-Esteban et al., 1998). Since correct cell-cell and cell-ECM interactions have been shown to be pivotal for liver proliferation (Weinstein et al., 2001), an alternative role for Lhx2 may be in the regulation of adhesive interactions between epithelial and mesenchymal cells, and/or between cells and the ECM (Figure 7C). Support for this hypothesis comes from functional studies of the Lhx2 ortholog apterous. During Drosophila wing development apterous regulates the establishment of dorsal and ventral compartments in the developing wing disc. apterous has been shown to maintain the dorsal-ventral boundary by regulating the adhesive properties of the cells within the different compartments (Blair et al., 1994). It has also been suggested that apterous control neuronal target recognition in Drosophila by regulating cell-surface properties (Lundgren et al., 1995). The function of Lhx2 in mature hepatic stellate cells in the

Figure 7. Model of the role of Lhx2 in early liver development. (A) Just after hepatic commitment Lhx2 is expressed in the st mesenchyme that lines the forming liver bud. (B) As the hepatic cords migrate out into the st mesenchyme Lhx2+ mesenchymal cells intermingle with the hepatoblasts. (C) During the following expansion of the nascent liver Lhx2 plays an essential role possibly by activating an exocrine signal that induces proliferation in the adjacent hepatoblasts (1) or by ensuring the correct adhesion between cells and/or cell and ECM, by for example negatively regulating ECM production (2).

35 RESULTS AND DISCUSSION adult liver may be distinct from the role of Lhx2 at earlier stages of hepatic development. However, the expression of Lhx2 in stellate cells provides a mechanism by which Lhx2 may affect adhesion. One of the major functions of hepatic stellate cells is production and remodeling of ECM (Friedman, 1999). Studies of E15 Lhx2-/- embryos have shown that their livers contain a large number of activated hepatic stellate cells and display elevated deposition of ECM (Wandzioch et al., manuscript submitted). Lhx2 thus appears to be required in hepatic stellate cells to negatively regulate their activation and ECM protein production. Assuming that the st mesenchymal cells that will generate hepatic stellate cells start to produce ECM at an early developmental stage, Lhx2 could affect cell adhesion during early liver development by negatively regulating their ECM production.

Lhx2 AND HEMATOPOIESIS (paper II)

Lhx2 immortalizes multipotent hematopoietic progenitor cells From E11 until just before birth, the liver serves as the main hematopoietic organ and the liver environment supports the maintenance and expansion of the entire HS including the HSCs (Ema and Nakauchi, 2000). Since the mesenchymal defect (paper I) in the liver of Lhx2-/- mice also leads to lethal anemia, indicating that the Lhx2-deficient liver microenvironment is unable to support hematopoiesis (Porter et al., 1997), Lhx2 could have a role in the maintenance and/or expansion of the HS. To examine a putative role for Lhx2 in fetal liver hematopoiesis, we took advantage of the ES in vitro system which, upon differentiation, recapitulates the earliest stages of hematopoietic development in the embryo (Keller et al., 1993). By introducing Lhx2 cDNA into ES cells and subjecting them to differentiation, we were able to test whether Lhx2 expression affects hematopoietic progenitor cells (Figure 8). Intriguingly, the EBs from Lhx2-transduced ES cells formed colonies containing multipotent hematopoietic progenitor cells. These colonies were named hematopoietic progenitor cell (HPC) colonies and were not observed in EBs from control-transduced ES cells. Cell lines, named HPC lines, could be established

36 RESULTS AND DISCUSSION

from the HPC colonies by transferring them to liquid culture. The HPC lines were strictly dependent on SF for efficient expansion and maintenance, and could be kept in culture for several months without losing their immature phenotype. In addition, individual HPCs exhibited a broad hematopoietic potential and could be efficiently induced to differentiate into most mature myeloid lineages in response to hematopoietic GFs. These features of the HPCs resemble both self-renewal and differentiation occurring in the hematopoietic system in vivo. Further characterization of the HPCs revealed that they expressed a number of transcription factors implicated in hematopoietic development and associated with normal HSCs, such as SCL, PU.1 and GATA1 (Scott et al., 1994; Weiss et al., 1994; Shivdasani et al., 1995). In addition, the pattern of cell-surface marker expression on the HPCs (c-kit+, CD34+, CD44+, Sca1-, Thy1.2- AA4.1neg/low, Lin-) was very similar to the profile of multipotent hematopoietic progenitor/stem cells isolated from E9 yolk sac (Yoder et al., 1997) or E11 fetal liver (Sanchez et al., 1996). Thus

Figure 8. Schematic overview of the establishment of the HPCs. ES cells were transduced with Lhx2 cDNA or control vector and differentiated into EBs. When the precursor content of the EBs generated from Lhx2 transduced ES cells was analyzed in clonal assays they were found to contain progenitors that generated a type of colonies never obtained form non-transduced EBs when replated in the presence of erythropoietin (Epo) and steel factor (SF). These colonies contained immature blast like cells and had the ability to generate additional blast cell colonies upon serial replating. Based upon these features the colonies were referred to as hematopoietic progenitor cell (HPC) colonies. In the presence of SF the HPCs could be kept in liquid culture for several months without loosing their immature phenotype. In response to hematopoietic growth factors a large proportion of the HPCs generated multilineage colonies containing cells of several lineages such as erythrocytes, macrophages, megakaryocytes neutrophils and mast cells, which demonstrated that the HPCs were multipotent.

37 RESULTS AND DISCUSSION expression of Lhx2 in differentiating ES cells led to the immortalization of a multipotent hematopoietic progenitor cell sharing several characteristics with fetal multipotent hematopoietic progenitor/stem cells. That the immortalization of the HPCs was a direct effect of Lhx2 expression, and not due to genomic cDNA insertion site or other side effects, was confirmed by the introduction of antisense Lhx2 cDNA into HPCs (Pinto do O et al., 2001) and by the establishment of HPC lines with inducible Lhx2 expression (DoxHPC lines; Richter et al., manuscript in preparation). In both cases, downregulation of Lhx2 expression led to reduced proliferation and/or cell death and terminal differentiation. Collectively, these data provide evidence that expression of Lhx2 is required for self-renewal of the HPC lines.

Lhx2 may regulate HSC self-renewal in vivo The specificity by which Lhx2 immortalizes a multipotent SF-responsive hematopoietic progenitor is striking, considering that the EBs contain a heterogeneous population of SF-responsive progenitors (Keller et al., 1993). Moreover, when Lhx2 was introduced into adult BM-derived hematopoietic progenitor cells, only immature multipotent progenitor/stem cells were immortalized (Pinto do O et al., 2002). This could be due to the fact that the Lhx2- induced immortalization requires a specific cellular and molecular context exclusively present in immature hematopoietic progenitor/stem cells. Although the precise mechanism of Lhx2-induced immortalization remains elusive, studies of the growth requirements of HPCs have revealed that the mechanism is cell non- autonomous (Pinto do O et al., 2001), i.e. that Lhx2 regulates, directly or indirectly, some soluble mediator(s) involved in self-renewal of the HPCs, or that Lhx2 facilitates the activity of such mediator already expressed in normal HSCs. The Lhx2-dependent generation and maintenance of the HPCs suggest a role for Lhx2 in expansion and/or maintenance of the hematopoietic system in vivo. This putative role is most likely cell non-autonomous since Lhx2-/- HSCs are normal (Porter et al., 1997). Lhx2 would thus be part of the hematopoietic support provided by the fetal liver stroma. Hence, the Lhx2-expressing hepatic stellate cells may in addition to being important for the expansion of the hepatic endoderm be involved in the expansion of the HS. Based on the mechanism of Lhx2-induced immortalization of the HPCs, a possible role for Lhx2 in the hematopoietic

38 RESULTS AND DISCUSSION

Figure 9. Model of a possible role of Lhx2 in expansion of HSCs in the liver. The Lhx2 expressing hepatic stellate cells may support expansion of the HSCs by directly regulating the expression of some soluble mediator (1), by facilitating HSC response to soluble molecules secreted independently of Lhx2, perhaps by negatively regulating ECM production (2) or by affecting hepatocytes (3) or other cells present in the liver (4) that in turn promotes expansion of HSCs.

microenvironment could be to directly or indirectly regulate the expression of soluble mediators involved in the expansion of the hematopoietic system (Figure 9). Alternatively, expression of Lhx2 may facilitate the hematopoietic cells to respond to soluble factors secreted independently of Lhx2.

Dkk-1, A PUTATIVE Lhx2-REGULATED GENE IN THE HEMATOPOIETIC AND HEPATIC SYSTEMS (paper III)

To further elucidate the role of Lhx2 in the context of the fetal liver, we attempted to identify Lhx2 downstream target genes. For this purpose, the HPC line offered an excellent tool, as the HPCs express high levels of Lhx2 (paper II) and can be expanded in vitro to generate an unlimited supply of material. The approach, which was based on cDNA subtraction, was set up to generate both genes putatively regulated by Lhx2 and hematopoiesis-associated genes specifically expressed in the HPC lines, presumably representing stem cell specific genes. To determine whether the isolated genes were putative Lhx2 targets and not upregulated in the HPCs independently of Lhx2, we used DoxHPC cell lines (Richter et al., manuscript in preparation) where the expression of Lhx2 depends on the presence of doxycycline (dox). Upon removal of dox from the culture medium, the promoter is turned off and Lhx2 expression reduced by 95% within 24 h. Expression analysis of the isolated genes revealed that the soluble antagonist of Wnt/β-catenin

39 RESULTS AND DISCUSSION signaling dickkopf-1 (Dkk-1), the transcription activator Egr3 and the mesoderm specific transcript 1 (Mest1) were rapidly downregulated upon removal of dox. To examine whether these putative targets were linked to Lhx2 expression during liver development, we next analyzed their expression in the fetal liver. Whereas Egr3 and Mest1 showed no distinct expression pattern in the liver, Dkk-1 was expressed in the st mesenchyme from around E10, and thereafter in the mesenchymal layer of cells that surrounds the liver lobes. Faint expression in cells scattered within the lobes could be detected using high-sensitivity radiolabeled in situ hybridization. That the expression pattern of Dkk-1 in the developing liver appeared to overlap with that of Lhx2 suggested that Dkk-1 might be a mediator of Lhx2 function in the liver context. Based on this observation and the fact that Dkk-1 is a secreted molecule, we decided to focus our studies on Dkk-1. When the expression of Dkk-1 was examined in Lhx2-/- embryos, we found that the level of Dkk-1 transcripts in the liver was significantly reduced as compared to the wild-type level. Normal expression levels of Dkk-1 in the liver are thus dependent upon functional Lhx2. These results further suggest a link between Dkk-1 and Lhx2 function in the liver. The fact that Dkk-1 is an extrinsic factor is in agreement with our model for Lhx2 mediated function in both liver expansion (paper I) and HPC self-renewal (Pinto do O et al., 2001; paper II).

Dkk-1 alone cannot mediate HPC self-renewal The soluble nature of Dkk-1 prompted us to examine whether Dkk-1 could be part of the Lhx2-induced HPC self-renewal (Pinto do et al., 2001). The ability of Dkk-1 to compensate for downregulated Lhx2 expression was tested by adding soluble Dkk-1 to DoxHPC cultures upon dox removal. Preliminary results indicated that Dkk-1 alone is unable to mediate self-renewal in the HPCs (data not shown). This does not, however, exclude a role for Dkk-1 in the Lhx2-induced immortalization of HPC lines. Additional Lhx2-regulated targets may be required to enable the HPCs to respond to Dkk-1. Identification of downstream events of other TFs, such as c-Myc for example (Zeller et al., 2003), has generated extensive numbers of target genes. It is therefore likely that Lhx2 activates or represses the expression of several different genes that act in conjunction to mediate Lhx2-induced activity. Support for a role for Dkk-1 in regulation of stem cells has come from the specific expression of Dkk-1 in human HSCs (Gomes et al., 2001) and from the

40 RESULTS AND DISCUSSION

ability of Dkk-1 to promote self-renewal of cultured bone marrow-derived mesenchymal stem cells (hMSCs) (Gregory et al., 2003). These observations together with the expression of Dkk-1 in the fetal liver environment at the time of active hematopoiesis suggest that Dkk-1 may be involved in the expansion of fetal liver hematopoiesis.

Dkk-1 acts via a Wnt/β-catenin independent mechanism in the liver Since Lhx2 appears to directly or indirectly regulate the expression of Dkk-1 in the developing liver and Dkk-1 thus could be part of Lhx2-induced liver proliferation (paper I), we were interested in understanding the role of Dkk-1 during liver development. As Dkk-1 is defined as a Wnt/β-catenin antagonist, we started by analyzing the activity of canonical Wnt signaling in the liver. To examine activation of the Wnt receptor at various stages of liver development, we used the BAT-gal Wnt reporter mouse (kindly provided by Professor Stefano Piccolo), where expression of the lacZ gene is under the control of β-catenin/TCF responsive elements (Maretto et al., 2003). As suggested from previous observations, there was an almost complete absence of active Wnt/β-catenin signaling in E9.5-E14 livers (Monga et al., 2003)(Figure 10A). Nor was any active Wnt signaling

Figure 10. Activation of the Wnt/β-catenin pathway in wild type and Lhx2-/- fetal livers. X- gal staining (blue) of E10.5 liver sections from Lhx2+/+ BAT-gal (A) and Lhx2-/- BAT-gal (B) mice. Co-staining of liver hepatocytes with an anti albumin antibody (brown). Note the almost complete absence of Wnt/β-catenin active cells (blue) in both wt and mutant livers.

41 RESULTS AND DISCUSSION detected in tissues surrounding the liver. If inhibition of the Wnt/β-catenin pathway is the main function of Dkk-1 in the liver, one would expect that the Lhx2-/- liver, with lower levels of Dkk-1 expression as compared to wild-type liver, would have increased levels of Wnt/β-catenin signaling. By crossing Lhx2+/- mice with the BAT-gal Wnt reporter mouse (Maretto et al., 2003) to generate Lhx2-/- reporter mice, the levels of Wnt/β-catenin signaling in wild-type and mutant livers could be compared. No obvious increase in Wnt/β-catenin signaling in the Lhx2 mutant was observed (Figure 10B), suggesting that the activity of Dkk-1 in the liver does not involve inhibition of β-catenin-dependent transcription. Even though Dkk-1 was originally defined as a Wnt/β-catenin inhibitor, there is some evidence of alternative Dkk-1 activity (Figure 11). Dkk-1 has, for example, been shown to activate the Wnt/c-Jun NH2-terminal kinase (JNK) signaling pathway during heart

Figure 11. Dkk-1 and the Wnt signaling pathways. (A) The Wnt/β-catenin pathway. Dkk-1 inhibits the Wnt/β-catenin (canonical) [reviewed in (He et al., 2004)] pathway by interacting with the Wnt co-receptors LRP5 or LRP6 and thereby blocking the formation of an active Wnt receptor complex (Bafico et al., 2001; Mao et al., 2001; Semenov et al., 2001). In the absence of active Wnt signal β-catenin is phosphorylated and targeted for degradation by the APC/Gsk3β/Axin complex. The level of cytosolic β-catenin and β-catenin/Tcf driven transcription of Wnt target genes is thereby kept low. Dkk-1 also interacts with Kremen1 and Kremen2 (Mao et al., 2002). (B) The non-canonical Wnt pathways. It has been indicated that Dkk-1 may activate non-canonical Wnt signaling (Pandur et al., 2002). How this may occur and whether such activation involves binding to LRP5 is not known.

42 RESULTS AND DISCUSSION development in Xenopus (Pandur et al., 2002). Moreover, the Dkk-1 receptor LRP5 has been postulated to functionally modulate JNK signaling by sequestering activated JNK into the plasma membrane and inhibiting activation of JNK- dependent TFs in the nucleus (Lutz et al., 2002), further indicating a possible link between Dkk-1 and JNK signaling. Though the eventual connection between Dkk- 1 and non-canonical Wnt signaling needs further investigation, our results suggest that if Dkk-1 mediates Lhx2-induced liver proliferation, it occurs independently of the Wnt/β-catenin pathway.

Lhx2 AND THE OLFACTORY SYSTEM (paper IV)

During the generation of the olfactory epithelium, neuronal progenitors differentiate into mature OSNs which are individually specified into over a thousand different neuronal subpopulations. Depending on their spatial position in the OE, the neurons are restricted to expressing ORs from a region-specific set of receptors and will project their axons to a corresponding region in the olfactory bulb. The mechanisms regulating OSN differentiation and diversification are not well understood. Although a few TFs belonging to the bHLH TF family have been implicated in olfactory neurogenesis (Guillemot et al., 1993; Cau et al., 1997), it is not known how differentiation, OR gene selection and zonal specification is regulated. Since Lhx2 has been implicated in neurogenesis in the developing forebrain and neural retina (Porter et al., 1997; Monuki E.S. et al., 2001), the expression of Lhx2 in the OE (Rincon-Limas et al., 1999)(Figure 12) suggested that Lhx2 could be involved in the differentiation and/or diversification of the OSNs.

Figure 12. Lhx2 expression in OE Coronal sections of an adult (A) and E14.5 (B) OE hybridized with an Lhx2 antisense RNA probe. In the adult OE Lhx2 shows a differential pattern of expression with high levels in the basal progenitor layer and lower expression in the mature neurons (A). The layered expression is not as evident during embryogenesis (B). No expression of Lhx2 is detected in the sustentacular cell layer or in cells beneath the OE.

43 RESULTS AND DISCUSSION

Lhx2 is required for the expression of OR genes and other markers of mature OSNs In order to analyze a potential role for Lhx2 in the OSNs, OE from E15 Lhx2 -/- and wild-type mice were compared. No obvious difference in thickness, cell layer organization and distribution of neuronal progenitors normally expressing Lhx2 was detected. However, when we analyzed the expression of odorant receptors in OSNs, we found that the Lhx2-/- OE completely lacked OR gene expression. Using high-sensitivity radiolabeled in situ hybridization, no OR gene expression was detected in any of the OE zones. These results strongly indicate that Lhx2 is required, directly or indirectly, for OR gene expression. In addition, OSNs in Lhx2- deficient embryos lacked expression of other zone-specific genes, i.e. NCAM2 and NQO1. Furthermore, the expression of markers for terminally differentiated OSNs, Gαolf and OMP, was significantly reduced and only detected in a few OSNs in Lhx2-/- OE as compared to wild-type OE. The lack of OR, NCAM2 and NQO1 expression in combination with reduced Gαolf and OMP expression indicated that Lhx2-deficient neurons fail to develop into functional OSNs.

Figure 13. Schematic picture of OE in wild type and Lhx2-/- embryos. In the absence of functional Lhx2 OSN differentiation is arrested after initiation of differentiation but before onset of OR expression. The OE displays an accumulation of immature OSNs that have exited cell cycle, express NeuroD and the pan neuronal markers Tubulin III, NCAM1, Gap43 and SCG10 and have started to project axons towards the bulb. The dorsomedial zone in the mutant OE contain a few OSNs that express markers for terminal differentiation such as OMP and Gαolf but lack OR gene expression.

44 RESULTS AND DISCUSSION

Accumulation of immature neurons in Lhx2-/- embryos In order to define at what stage of OSN differentiation and diversification Lhx2 function is critical, we followed the differentiation of olfactory neuronal progenitors in the Lhx2-/- OE. The generation of neuronal progenitors and early neurogenesis appeared to proceed normally in the absence of functional Lhx2, as detected by unaltered expression of Mash1 and Ngn1 (expression data summarized in Figure 13). However, in the absence of Lhx2, an accumulation of NeuroD and Tubulin III co-expressing neurons, i.e. neurons that had left the cell cycle and initiated differentiation, was detected. The neurons in the OE of Lhx2-/- mice acquired other pan-neuronal characters as well, such as NCAM1, Gap43 and SCG10, and started to project their axons towards the olfactory bulb. These results suggested that neuronal differentiation in the absence of Lhx2 is arrested at a stage at which the neurons have initiated terminal differentiation, but before the induction of OR expression.

Lhx2 and regional specification Perhaps the most intriguing consequence of disrupted Lhx2 expression in the OE was a zone-specific difference in neuronal maturation. The few neurons that expressed late differentiation markers, such as OMP and Gαolf, and acquired an OSN identity in the absence of functional Lhx2, were exclusively located in the dorsomedial region – whereas the ventrolateral region of the Lhx2-/- OE contained a higher fraction of newly differentiated post-mitotic NeuroD positive cells. The absence of Lhx2 thus revealed zone-specific differences in the regulation of OSN differentiation that are normally unapparent. Some zonal differences in OSN maturation have been described under normal conditions as well (Iwema and Schwob, 2003). While OSNs normally downregulate expression of Gap43 prior to onset of OMP expression, OSNs in the dorsomedial region initiate OMP expression independently of Gap43 inactivation. The dorsal OSNs thus appear to be less sensitive to differentiation checkpoints than OSNs in the rest of the epithelium, which could explain why the dorsal OSNs in the Lhx2-/- OE express late differentiation markers such as OMP and Gαolf, despite a lack of OR gene expression Alternatively, the zonal difference in neuronal differentiation could be a consequence of disrupted Lhx2 expression in OSN progenitors. The discovery that Rhombotin 1 (Rbtn1 or LMO1) is exclusively

45 RESULTS AND DISCUSSION expressed in basal progenitors and immature olfactory neurons in the ventrolateral zone suggested that Lhx2 could be involved in zonal specification (Tietjen et al., 2003). Rbtn1 can interact with LIM-HD factors via the LIM interactor NLI (Jurata et al., 1998) and might thereby modify the transcriptional activity of Lhx2 in a zonal manner. While manuscript IV was being submitted, another paper describing OSN differentiation in the absence of Lhx2 was published (Hirota and Mombaerts, 2004). Although these authors did not report any OMP or Gαolf expression, and thus no zonal differences in OSN differentiation in the Lhx2-/- OE, they found, in agreement with our results, that Lhx2 is required for OSN development.

Lhx2 - a putative regulator of OR gene expression With regard to the lack of OR gene expression in the mutants, it is interesting to note that Lhx2 has been identified in two separate screenings for candidate TFs that directly regulate OR gene transcription (Hoppe et al., 2003; Hirota and Mombaerts, 2004). In addition, several OR promoters belonging to different OR gene clusters have been shown to contain homeodomain binding sites located both at a distance to and in close proximity to the transcriptional start site (Hoppe et al., 2000; Lane et al., 2001; Vassalli et al., 2002). This indicates that HD TFs may be involved in different aspects of OR gene regulation. Although Lhx2 has been shown to physically interact with the promoters of two specific OR genes, M71 and mOR262-6 (Hoppe et al., 2003; Hirota and Mombaerts, 2004), the complete lack of OR gene expression in the Lhx2-/- mice (paper IV and (Hirota and Mombaerts, 2004) suggests that Lhx2 functions upstream of the mechanism that ensures expression of one defined OR gene per neuron. The differentiation block observed in the Lhx2-/- OSNs may coincide with a previously described delay between onset of pan-neuronal gene expression and OR gene expression (Iwema and Schwob, 2003). Interestingly, accelerated differentiation in response to onset of OR expression has been proposed to be a way of restricting the number of OR genes expressed in a given cell (Serizawa et al., 2003; Lewcock and Reed, 2004). In line with the suggested model, in which the OR proteins themselves initiate a negative regulation signal, the delay may correspond to the time required for synthesis of OR protein until a feedback signal can be generated. If selection of OR gene expression is a prerequisite for the OSNs

46 RESULTS AND DISCUSSION to proceed to the next step in development, Lhx2-/- OSNs would be unable to progress past this stage of selection since functional Lhx2 appears to be required for OR gene expression. This would thus explain the accumulation of immature OSNs in the Lhx2 mutant. Even though the above-mentioned data support a role for Lhx2 in regulation of OR gene expression, functional data demonstrating that binding of Lhx2 to the OR promoters is required for OR gene expression are essential for elucidation of whether the requirement of Lhx2 for OR gene expression is direct or indirect.

A role for Lhx2 in olfactory progenitor cells? Although the Lhx2 mutant phenotype could be explained at least in part by a role for Lhx2 in regulation of OR gene expression, the defective OSN differentiation may just as well be due to deficient Lhx2 function at an earlier differentiation/ progenitor stage. The lack of OR gene expression could be due to that progenitors fail to develop into the stage where OR gene expression is initiated or into cells capable of OR gene expression. At present, the only regulators expressed in basal progenitors that are implicated in OSN neurogenesis are the bHLH TFs Mash1, Ngn1 and NeuroD (Guillemot et al., 1993; Cau et al., 1997; Cau et al., 2002). The unaltered expression of Mash1 and Ngn1 in the Lhx2 mutant OE and the unaffected Lhx2 expression in Ngn1-/- OE (Cau et al., 2002) suggest that if Lhx2 has a role in OSN progenitors, it is most likely not part of the bHLH pathway. The differentiation of OSNs may be regulated by two independent pathways, in a manner similar to the developing retina where the regulators Pax6 and Lhx2 act independently of each other through parallel and equally important pathways (Porter et al., 1997). Although the precise mechanism of Lhx2 function remains to be elucidated, we can conclude that Lhx2 is intrinsically required in the OSN lineage for the generation of a heterogeneous population of individually and zonally specified OSNs.

47 RESULTS AND DISCUSSION

SUMMARY OF RESULTS AND DISCUSSION

The work presented in this thesis addresses the role of the LIM-HD transcription factor Lhx2 during development of the liver, expansion of the hematopoietic system and differentiation/specification of olfactory sensory neurons. Our findings provide additional evidence for the ability of Lhx2 to trigger a variety of cellular responses in different organ systems via both cell autonomous and cell non- autonomous mechanisms. During liver development, Lhx2 is expressed in the hepatic mesenchyme that will generate hepatic stellate cells and appears to affect both the expansion of the liver and the hematopoietic system via cell non-autonomous mechanisms. From early stages of liver development, the Lhx2 expressing st mesenchymal cells are in close contact with both hepatoblasts and hematopoietic cells. Disruption of Lhx2 expression in these cells leads to en early liver expansion defect, which indicates that expression of Lhx2 in the hepatic mesenchyme is required for the expansion of adjacent hepatoblasts. The ability of Lhx2 to immortalize hematopoietic progenitor cells together with the lethal anemia in Lhx2-/- mice suggest that Lhx2 expression in the hepatic mesenchyme also is required for maintenance and expansion of the HS. The molecular basis of these Lhx2-mediated activities is not fully understood at present, but we suggest that they may involve the production of some exocrine molecule, possibly the soluble Wnt inhibitor Dkk-1, that acts on the adjacent hematopoietic and/or hepatic cells as well as the regulation of adhesive molecules/ECM required for correct cell-cell interactions. Our findings suggest that in the hepatic mesenchyme, Lhx2 plays a pivotal role in cell-cell interactions. During the differentiation and diversification of olfactory sensory neurons expression of Lhx2 is required in the OSN lineage for the generation of bona fide OR-expressing neurons. A possible role for Lhx2 in OSN development is in the regulation of OR gene expression, as OSNs in the absence of functional Lhx2 fail to express OR genes, and Lhx2 binding sites have been identified in OR gene promoters. Alternatively, Lhx2 is required in olfactory progenitors at an early developmental stage for them to develop into cells capable of OR expression. Though the exact mechanism of Lhx2 function in the OSNs remains unsolved our findings suggest that Lhx2 acts via a cell autonomous mechanism to regulate the differentiation and specification of olfactory sensory neurons.

48 CONCLUSIONS

CONCLUSIONS

• Lhx2 is expressed in liver associated septum transversum mesenchyme that becomes integrated in the developing liver and generates a population of hepatic stellate cells. Expression of Lhx2 in the mesenchyme is required for the mesenchymal-epithelial interactions that control liver expansion.

• Expression of Lhx2 in ES cells differentiated in vitro leads to the generation of immortalized multipotent hematopoietic progenitor cells that self-renew and differentiate in vitro and hence share several characteristics with hematopoietic progenitor/stem cells. The establishment of these HSC- like cell lines together with the lethal anemia in Lhx2-/- mice suggests a role for Lhx2 in expansion/maintenance of hematopoiesis in vivo.

• The soluble Wnt/β-catenin signaling antagonist Dickkopf-1 is putatively regulated by Lhx2 in the fetal liver context and may contribute to the function of Lhx2 in the liver.

• Lhx2 is intrinsically required in olfactory neuron progenitors for differentiation and generation of individually specified mature olfactory sensory neurons

49 ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

There are a number of people that I specifically would like to give my gratitude and appreciation to.

First of all I would like to thank my supervisor Leif Carlsson, for taking me in to the lab and giving me the opportunity to carry out this thesis, for always having the time and energy to discuss work and theories and for improving my skills in fighting for my ideas. Thanks for all your support.

All the former and present members of group LC. I can’t imagine a better bunch of people to work and spend time with every day. Ewa, for sharing projects, lab space, high flying scientific speculations, thoughts about life in general and how to become rich in particular, and many, many laughters. And, for being a friend. Live long and prosper! Karin, for adding flavor to everyday lab work, for your smiles, for being such a loving person and for almost never saying no to a coffee break. Perpétua, for the good times we had in the tiny lab at CMB, when the group was just you and me(and Leif), for growing together as scientists – although I could never keep up with you, and for teaching me that it is OK to eat a whole box of ice cream in one evening. AnnaCarin, for your cool attitude, for coping with all my talking and for your “luriga” comments. Christofer, for being the rooster in the hen house and the cutest Lucia ever. We really miss you! Gunilla, for bringing new energy to the lab and joining the “cirkelteam”. Maria, for together with Gunilla forcing us “oldies” to have some fun. Ludmilla, for the help with screening the subtraction clones and for your enthusiasm for everything from gardening to Swedish classes. And all of you who have brought color to the lab, if only for shorter periods of time, especially Elisabet, Lars, Maria N and Caroline for new influences and good company.

During the course of my PhD I have had the opportunity to work at two different departments, Molecular biology and UCMM. Two places filled with excellent scientists, helpful colleagues and great friends. Even though it feels like a long time since we moved from Molbiol it always feels good going back there and being greeted with a “Tjenare Åsa, det var länge sen…” I specifically want to thank all the fantastic people at UCMM for making it such a great working place. For giving lunch discussions concerning the being or not being of the “punghålet”, energy filling coffee breaks, Friday evenings in the beer corner, for always giving a helping hand and, most importantly, for great friendship.

Staffan Bohm for being so enthusiastic and for your ability to find something of interest even in the experiments that didn’t turn out the way they were supposed to. It has been a pleasure working with you and having you as guide in the olfactory field.

Anna & Staffan and past and present members of their groups; Mattias, Marianne, Fredrik, Maria, Viktoria, Marie, Björn, and Lennart for keeping a welcoming atmosphere in your lab, for helping me with radio-labeled in situ, and for patiently answering questions and being good listeners.

50 ACKNOWLEDGEMENTS

Marita Waern, Kristina Strindlund and Anita Bergdahl for excellent guiding through the administrative jungle, and for always having the time and the answers. P-A Lundström, for solving the ordering problems with ease, for being a reliable source of gossip, and for making everyone in the department feel that they belong. No one is spared from your comments. The ladies at disken and media deserves special thanks for making life in the lab so much easier and for always being open for weird special wishes. Elisabeth Nilsson at the animal facility, for excellent work, for keeping track when I’m lost and for spoiling me with deliveries at the door.

Anna M., Lelle, Laila, Yngve, Anna F. and Mattias for our monthly lunches, and for sharing the frustrations and enjoyments of being (or having been) a PhD student.

Lelle, Gussing, Anna-Lena and Ewa, for sharing the experience of thesis writing and for being great company at a summer empty department. A special thanks to Lelle, who has been the best company, coach, support and “jägarsnus” supplier any one could ever wish for during their thesis writing.

Anna F, for your friendship, for sharing thoughts about what’s important in life and for leading the way. I missed having “lassaparks” lunch dates with you this summer.

Mattias, for being a great friend, for long phone calls and for enjoyable and fruitful collaboration on the OE project.

Sus, for being the kind of friend I wish everyone would have, for calling me on Thursday evenings to say that you saved me a seat in the car to Kittelfjäll and for knowing that you always will be there, no matter what.

Till mina vänner i bönegruppen, där jag fått hämta både vila och kraft, för att ni har erbjudit ett annat perspektiv på tillvaron än det som finns på lab. Tack för alla stunder av både allvar och skratt.

Till alla goda vänner- för att ni alltid finns där. Vad vore livet utan er! Tack för allt kul vi har haft, alla fikan och afterworks, utflykter och iksuträffar och för den särskilda omtanke ni visat sen Jonas åkt.

Bengt & Anita Persson, Sara & Henri, tack för omtanke och vänskap och för att ni får oss att känna oss som hemma på Talludden.

Maria & Anders, Oskar och Sara, för vänskap, peppning och nattlig Word support.

Mamma & pappa, Eva & Björn, för er kravlösa kärlek, omsorg och tro på mig. Och för vetskapen att ni alltid finns där.

Jenny & Martin, för allt stöd och alla pep-talks, för nära vänskap och för sköna avkopplande Göteborgsbesök.

Jonas, du är mitt allt. Utan ditt stöd, lugn och din kärlek skulle jag inte vara den jag är. Jag ser så fram emot att snart få dela dagarna med dig igen.

51 REFERENCES

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