Aus dem Institut für Anatomie und Zellbiologie Abteilung Anatomie II der Albert-Ludwigs-Universität Freiburg im Breisgau und From the Department of Biochemistry and Molecular Biology MD Anderson Cancer Center, Houston, Texas, USA

Detailed analysis of Lmx1b expression in multiple organs and its role in dorso-ventral patterning with emphasis on myogenic precursor cells

INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades

der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg im Breisgau

Vorgelegt 2003 von Heiko Schweizer geboren in Freiburg im Breisgau

Dekan: Prof. Dr. Josef Zentner Erstgutachter: Prof. Dr. Beate Brand-Saberi Zweitgutachter: Prof. Dr. Randy Johnson Jahr der Promotion: 2005

In Gedenken meiner lieben Mutter gewidmet

Table of Contents

A1 GERMAN SUMMARY (DEUTSCHE ZUSAMMENFASSUNG) ...... 1

A2 ENGLISH SUMMARY...... 2

1. INTRODUCTION ...... 3

1.1 Lmx1b – a LIM homeodomain transcription factor...... 4 1.1.1 Structure and properties of Lmx1b...... 4 1.1.2 Expression of Lmx1b in mouse and chick ...... 5 1.1.3 Effects of gain-of-function and loss-of-function of Lmx1b...... 7 1.1.4 Lmx1b is involved in the Nail-patella syndrome (NPS) ...... 8 1.1.5 Lmx1b expression is affected in transgenic mice and by overexpression of other limb genes...... 9 1.1.6 Downstream targets of Lmx1b...... 10 1.1.7 Induction of Lmx1b ...... 10

1.2 General limb development...... 12 1.2.1 Axis formation ...... 13 1.2.2 The proximo-distal axis of the limb ...... 14 1.2.3 The anterior-posterior axis of the limb ...... 15 1.2.4 The dorso-ventral axis of the limb ...... 15 1.2.5 Genetic interactions along the three axes ...... 17 1.2.6 Establishment of the dorso-ventral boundary and boundaries in general...... 18 1.2.7 Muscle formation ...... 20

1.3 Mammary gland development ...... 26 1.3.1 Fetal development ...... 26 1.3.2 Postnatal development...... 27 1.3.3 Pubertal and postpubertal development...... 27 1.3.4 Adult development including pregnancy and involution...... 27 1.3.5 The cellular composition of mammary glands ...... 28

1.4 Kidney development...... 29 1.4.1 WT-1: Wilms’ tumor suppressor gene...... 29

2. MATERIALS AND METHODS...... 31

2.1 Animals...... 31 2.1.1 Mice ...... 31 2.1.2 Chick and quail ...... 32

2.2 Microsurgery and interspecific grafting experiments...... 33 2.2.1 Isolation of mouse embryos ...... 33 2.2.2 Dissection of mammary glands ...... 33 2.2.3 Quail to chick grafting experiments ...... 34

2.3 Cell culture and in vitro culture of embryos...... 40 2.3.1 Cell culture ...... 40 2.3.2 Micromass culture...... 43 2.3.3 Whole embryo culture...... 46

2.4 Molecular biology...... 48 2.4.1 General methods...... 48 2.4.2 Large plasmid DNA preparation ...... 49 2.4.3 Preparation of digoxygenin labeled probes ...... 50 2.4.4 Genotyping of transgenic mice by Southern blotting and PCR...... 51

2.5 Whole mount and histological analysis...... 56 2.5.1 X-gal staining ...... 56 2.5.2 Immunohistochemistry...... 61 2.5.3 In situ hybridization histochemistry...... 64

2.6 Photography and imaging...... 68

2.7 Solutions, buffers and antibodies ...... 69

3. RESULTS...... 72

3.1 Lmx1b 3’LacZ mice ...... 72 3.1.1 Genotyping...... 72 3.1.2 Phenotype, expression level and localization...... 73 3.1.3 Intercrosses of Lmx1b 3’LacZ/LacZ X Lmx1b KO +/- mice...... 74

3.2 Detailed analysis of Lmx1b expression in Lmx1b 3’LacZ transgenic mice ...... 75 3.2.1 Expression of Lmx1b in the murine limb ...... 75 3.2.2 Expression of Lmx1b during mammary gland development ...... 80 3.2.3 Lmx1b expression in the kidney ...... 84 3.2.4 Lmx1b expression in other organs and in adults...... 85 3.2.5 Brief summary of Lmx1b expression...... 87

3.3 Induction of Lmx1b and boundary formation ...... 88 3.3.1 Lmx1b positive cells maintain a homogenous distribution in a micromass culture...... 88 3.3.2 Ventral proximal mouse limb mesenchyme does not initiate Lmx1b expression when grafted ...... into the Progress Zone...... 89 3.3.3 No induction of Lmx1b in proximal ventral cells by Wnt7a and FGF8b in the limb ...... 89 3.3.4 Lmx1 - induction of ventral proximal cells in chick...... 90

3.4 Migration behavior of grafted quail cells in the chick wing ...... 91 3.4.1 Lbx1 expression in quail and chick ...... 91 3.4.2 Interspecific grafting experiments...... 92 3.4.3 Brief summary of grafting experiments ...... 98

4. DISCUSSION...... 99

4.1 Analysis of LacZ reporter mice provides novel insight into the Lmx1b expression pattern ..... 99 4.1.1 Lessons from Lmx1b genetics ...... 99 4.1.2 Lmx1b detection in Lmx1b 3’LacZ reporter mice corresponds to known patterns ...... and reveals novel sites of expression...... 100 4.1.3 Lmx1b is expressed during all stages of mammary gland development...... 104 4.1.4 In the kidney, Lmx1b expression is restricted to podocytes and accounts for nephropathy ...... 106

4.2 A new perspective of the dorso-ventral boundary...... 108 4.2.1 The razor-sharp line of expression at the boundary does not respect anatomical structures ...... 108 4.2.2 Dorsal and ventral compartments are not maintained by repeling properties ...... of all mesenchyme cells ...... 108 4.2.3 Migrating cells respect the boundary between dorsal and ventral and do not change sides...... 111 4.2.4 Different myogenic lineages in the dorsal and ventral premuscle mass...... 112

4.3 Lmx1b expression with respect to the proximo-distal axis...... 114 4.3.1 Proximal cells maintain their identity permanently...... 114 4.3.2 The organizer region is not reestablished after dispersion of limb cells...... 116 4.3.3 Different migration behavior in proximal and distal limb regions ...... 117 4.3.4 Reverse proximal migration and myogenic attraction...... 119

4.4 Integration of different mechanistic models of limb axis formation with respect to...... Lmx1b expression ...... 121

5. REFERENCES ...... 125

6. APPENDIX ...... 165

6.1 Tables and Figures ...... 165

6.2 Abbreviations...... 166

6.3 Acknowledgements...... 167

6.4 Curriculum vitae ...... 168

6.5 Declaration (Erklärung über die Beteiligung Dritter) ...... 170

B FIGURES ...... 171

1 Summary

A1 German Summary (Deutsche Zusammenfassung) Das Lmx1b-Gen kodiert für einen Transkriptionsfaktor mit LIM- und Homöodomänen. Die Ausschaltung dieses Gens bei Mäusen führt zu biventralen Extremitäten und Defekten in Skelett, Gehirn, in der Niere und in den Augen (Chen et al. 1998a). Ähnliche Defekte sind beim humanen Nail-Patella-Syndrom (NPS) beschrieben. Die genaue Untersuchung der Expression von Lmx1b in Mäusen wurde durch Einfügung eines LacZ Reportergens ermöglicht, das vom endogenen Lmx1b Promotor kontrolliert wird. Diese Arbeit gewährt einen neuen Einblick in das Expressionsmuster dieses Gens, wobei auch bisher unbeschriebene Organe untersucht und die dortige Expression detailliert charakterisiert werden. So wird zum ersten Mal gezeigt, dass Lmx1b in allen Stadien der Brustdrüsenentwicklung exprimiert ist. Nur die luminal gelegenen Epithelzellen erweisen sich als positiv. In Lmx1b Knock-out Mäusen fehlen die Brustdrüsen vollständig. Diese Erkenntnisse weisen darauf hin, dass Lmx1b eine wichtige Rolle in der Brustdrüsenentwicklung spielt. Tatsächlich wurde bei Patientinnen mit NPS eine Minderentwicklung der Brust beschrieben (Sweeney et al. 2003). Des Weiteren wird gezeigt, dass das Gen in der Niere nur in den Podozyten exprimiert ist. Dies könnte mit der Nephropathie von NPS Patienten in Zusammenhang stehen. In Leber, Herz und Milz hingegen wurde keine Expression von Lmx1b gefunden. Außerdem wird gezeigt, dass Lmx1b in allen Stadien der Extremitätenentwicklung im dorsalen Teil exprimiert ist. Die bis zur Mitte reichende Expression grenzt die dorsale Seite scharf nach ventral ab. Zellkulturversuche mit gemischten dorsalen und ventralen Zellen ergaben, dass beide Populationen homogen verteilt bleiben und sich nicht aussortieren. Dies weist darauf hin, dass die scharfe Grenze nicht durch unterschiedliche Zelladhäsion aller Extremitätenzellen zu erklären ist, sondern vielleicht nur im Bereich der Grenze selbst besteht. Transplantationsexperimente zwischen Wachtel- und Hühnerembryonen zeigen, dass wandernde Zellen die dorso-ventrale Grenze nicht überschreiten. In die Extremität einwandernde myogene Vorläuferzellen haben sich als Lmx1b negativ erwiesen und vermutlich wird Lmx1b auch in differenzierten Muskelzellen nicht exprimiert. In dieser Arbeit werden zudem neue Wanderungswege und unterschiedliches Verhalten entlang der Grundachsen von Muskelvorläuferzellen beschrieben. Die Entdeckung von Lmx1b in weiteren Organen und die detaillierte Beschreibung seiner Expression stellt eine wichtige Grundlage für die weitere Forschung dar. Sie trägt dazu bei, das menschliche NPS besser zu verstehen und möglicherweise eines Tages behandeln zu können. 2 Summary

A2 English Summary Lmx1b belongs to a family of LIM homeodomain transcription factors expressed in multiple organs. Targeted disruption of Lmx1b results in ventralization of the limbs, skeletal, brain, kidney and ocular defects which resembles the corresponding human Nail- Patella Syndrome (NPS) (Chen et al. 1998a). The generation of transgenic mice carrying a LacZ reporter gene under the control of the endogenous Lmx1b locus allowed detailed analysis of Lmx1b expression. This study provides novel insights in the expression pattern, determines precisely Lmx1b-positive cell types in tissues, and describes for the first time expression in organs. It was demonstrated that Lmx1b expression is present throughout all stages of mammary gland development and restricted to luminal epithelial cells. Mice targeted for Lmx1b lack mammary glands. These findings indicate that Lmx1b plays a pivotal role in mammary gland development. Indeed, female patients with NPS display poor breast development (Sweeney et al. 2003). It is shown that Lmx1b expression is restricted to podocytes in the kidney, indicating the involvement of Lmx1b in nephropathy of NPS patients. No expression was detected in heart, liver and spleen. Lmx1b is involved in pattern formation of the limb and exhibits expression throughout all stages of embryonic development restricted to the dorsal limb mesenchyme. Hereby, Lmx1b expression marks a sharp boundary towards the ventral part of the limb. It is shown that dorsal Lmx1b-positive cells do not sort out from ventral Lmx1b-negative cells. This suggests that the maintenance of the boundary is not triggered by differential cell adhesion in the whole limb, maybe only at the boundary itself. Moreover, interspecific grafting experiments using quail-chick chimeras showed that migrating cells have restricted movement within the dorsal or ventral compartment they attain during transplantation. These findings indicate that migrating cells respect the boundary between the dorsal and ventral compartment and no exchange occurs. Interestingly, although Lmx1b is known as a marker for the dorsal limb mesenchyme, myogenic precursor cells invading the dorsal part of the limb are shown to be devoid of expression. It is also assumed that differentiated muscle lacks Lmx1b expression. In all, this study extends the understanding of limb pattern formation and reveals novel findings concerning proximo-distal differences and migratory pathways. The discovery of Lmx1b expression in organs and its detailed description forms an important basis for further investigations to finally understand and treat human NPS.

3 Introduction

1. Introduction

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“For the real amazement, if you wish to be amazed, is this process. You start out as a single cell derived from the coupling of a sperm and egg; this divides in two, then four, then eight, and so on, and at a certain stage there emerges a single cell which has as all its progeny the human brain. The mere existence of such a cell should be one of the great astonishments of the earth. People ought to be walking around all day, all through their waking hours calling to each other in endless wonderment, talking of nothing else except that cell.” (Lewis Thomas, 1979)

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“Thus, beyond all questions of quantity there lie questions of pattern which are essential for understanding Nature.” (Alfred North Whitehead, 1934)

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4 Introduction

1.1 Lmx1b – a LIM homeodomain transcription factor 1.1.1 Structure and properties of Lmx1b

Lmx1b, a LIM homeodomain transcription factor, was cloned first in hamster searching for genes regulating insulin expression (German et al. 1992). The location of Lmx1b was refined to the distal portion of the mouse chromosome 2, syntenic to the human region 9q34 where mutations in patients with Nail-Patella Syndrome have been detected (Campeau et al. 1995; Katsanis et al. 1996; Iannotti et al. 1997; McIntosh et al. 1997) The coding region of the murine Lmx1b gene contains 8 exons which encode for two amino-terminal LIM domains followed by a 60 amino acid homeodomain, a characteristic feature of the LIM homeodomain family (Sanchez-Garcia and Rabbitts 1994). “LIM” is derived from the names of the three LIM domain proteins: Lin-11 from Caenorhabditis elegans, Is1-1 from rat and Mec-3 from C.elegans. Each LIM domain binds two ions of Zn (II) forming two zinc fingers (Michelsen et al. 1993; Archer et al. 1994). The two cysteine-histidine rich LIM domains are followed by a homeodomain and a transcriptional activation domain facilitating interactions with other transcription factors (Xue et al. 1993; German et al. 1994; Curtiss and Heilig 1998). Investigation in a potential positive autoregulation of its own transcription did not show to stabilize the expression of Lmx1 (Riddle et al. 1995).

Figure I1.1: Phylogenetic tree illustrating the relationship of LIM homeodomain family members

5 Introduction

Members of the LIM family of transcription factors are frequently involved in pattern formation during development (Sanchez-Garcia and Rabbitts 1994; Curtiss and Heilig 1998), and appear to function as regulators of cell fate in different tissues (Tsuchida et al. 1994; Lundgren et al. 1995; Shawlot and Behringer 1995). Two homologues of the Lmx gene were cloned in hamster termed Lmx1.1 (German et al. 1992) and Lmx1.2 (Johnson et al. 1997) corresponding to the mouse orthologues Lmx1a (Millonig et al. 2000) and Lmx1b (Chen et al. 1998a) and to the human orthologues LMX1A (Thameem et al. 2002) and LMX1B (Dreyer et al. 1998; Vollrath et al. 1998) Up to now, only one Lmx1 gene has been isolated in chick (Riddle et al. 1995; Vogel et al. 1995).

1.1.2 Expression of Lmx1b in mouse and chick

1.1.2.1 Lmx1b expression in mice Most studies on Lmx1b expression were performed in chick and only few detailed examinations are available in mice (Cygan et al. 1997; Chen et al. 1998a; Chen et al. 1998b; Dreyer et al. 1998; Dreyer et al. 2000; Kania et al. 2000; Pressman et al. 2000; Smidt et al. 2000; Morello et al. 2001; Asbreuk et al. 2002; Miner et al. 2002; Rohr et al. 2002). First expression of Lmx1b was detected in the lateral plate mesoderm of the presumptive limb bud at E8.5, showing a restriction to the dorsal mesenchyme during outgrowth of the limb at E11.5 and remaining highly expressed in the dorsal autopod at E14.5 (Cygan et al. 1997). During eye development, Lmx1b was detected at E10.5 in the periocular mesenchyme, in the corneal stroma, surface ectoderm and later in the iris, cornea and trabecular meshwork (Pressman et al. 2000; Pressman 2001). Lmx1b is expressed in distinct areas of the brain including mesencephalic dopaminergic neurons at E16.5 and in adult humans (Smidt et al. 2000). The neuroepithelium underlying the developing skull and part of the cranial mesenchyme were also positive (Chen et al. 1998b). Lmx1b mutant mice lack mammary glands indicating that Lmx1b is involved in mammary gland development. In addition, no studies exist describing expression of the transcription factor in mammary glands. Thus, it was interesting to examine Lmx1b expression in mammary glands throughout all stages of development.

6 Introduction

First kidney expression was described by H. Chen (Chen et al. 1998a) in the S-shaped body of the metanephric kidney giving rise to the glomerulum. By E15.5, expression was detected in the mesenchyme surrounding the branching uteric tips and in glomerula where it persisted until birth. Patients with NPS with signs of a renal disease showed ultrastructural abnormalities of the glomerula basement membrane (GBM) (Taguchi et al. 1988; Dreyer et al. 1998).

1.1.2.2 Lmx1 expression in chick embryos

1.1.2.2.1 Lmx1 expression during early development Examination of Lmx1 expression and its genetic interactions in chick has been a challenging task for several research groups (Riddle et al. 1995; Vogel et al. 1995; Fernandez-Teran et al. 1997; Giraldez 1998; Yuan and Schoenwolf 1999; Adams et al. 2000; Mauch et al. 2000; Altabef and Tickle 2002) Yuan and Schoenwolf (Yuan and Schoenwolf 1999) described in detail early Lmx1 expression in chick: First transcripts were detected in the rostral midline mesoderm, the rostral tip of the primitive streak at stage 4. During neurulation, the neuroectoderm and epidermal ectodermal components of the neural folds and the elongating notochord were positive. After closure, the neural tube was stained in its entire length as well as the floor plate of the brain. Moreover, the midbrain and rostral hindbrain expressed Lmx1 as well as several other domains during and shortly after neurulation, including the prechordal plate and rostral head mesenchyme, foregut endoderm, otic placode and vesicle, dorsal somitic mesoderm, midline endoderm at the level of the caudal spinal cord, mesonephros and limb bud mesoderm. Giraldez and coworkers reported ectodermal expression during early somitic stages (4-6 somites), becoming restricted to the otic placode where expression persisted throughout otic cup and otic vesicle stages (Giraldez 1998). Additionally, few positive cells were mentioned in the dorsal portion of somites, but the data was not shown (Giraldez 1998). From HH stages 14-16, Lmx1 was expressed in the trunk dorsal ectoderm, the mesonephros, in the notochord and the presumptive gut endoderm. Neural tube expression was uniform at HH stage 14 and became restricted to the roof plate, floor plate and a subset of dorsal neurons later on (Riddle et al. 1995).

7 Introduction

Moreover, Lmx1 was detected in the isthmus organizer at the mesencephalic/metencephalic boundary (Adams et al. 2000). Lmx1 was expressed selectively in the dorsal limb bud mesenchyme (Dealy et al. 1993; Parr et al. 1993; Riddle et al. 1995; Vogel et al. 1995). Other members of the LIM family expressed in the limb including Isl1, Isl2 and LH2B were not restricted to the dorsal mesenchyme (Riddle et al. 1995). First expression of Lmx1 in the limb was detected at HH stage 15, being restricted at HH17 to the mesoderm adjacent to the dorsal ectoderm expressing Wnt7a (Riddle et al. 1995; Vogel et al. 1995). At HH stage 21, Lmx1 formed a sharp boundary at the border between dorsal and ventral which has only been described until HH stage 29. Cells undergoing the process of chondrification were reported to be devoid of Lmx1 expression (Riddle et al. 1995).

Only rare information is available about Lmx1 expression in the kidney of chick embryos. Mesonephric tubules expressed Lmx1 from stage HH16 on, while Pax2 was found in the mesonephric tubules, the nephric duct and its connecting segment. The pronephros did not reveal any expression (Fernandez-Teran et al. 1997; Giraldez 1998; Mauch et al. 2000).

While Lmx1 expression in chick has been examined in detail, the expression pattern in mice leaves many open fields for investigation. It remained unclear if other organs besides the kidney and the brain express Lmx1. One aim of this study was to screen murine tissues for Lmx1b and to describe up to now unpublished expression patterns.

1.1.3 Effects of gain-of-function and loss-of-function of Lmx1b

In a gain-of-function experiment, an RCAS-Lmx1 construct was injected into the limb primordium of stage 8-10 chick embryos, resulting in severe morphological alterations and double dorsal characteristics (Riddle et al. 1995; Vogel et al. 1995). Feet appeared hyperextended and planar, ventral muscles and tendons resembled closely the pattern of dorsal equivalents. Vogel and coworkers reported bidorsal feather patterns, abnormal or missing ventral structures, loss of ventral muscles or supernumerary dorsal muscles within the ventral side after ectopic expression of Lmx1 (Vogel et al. 1995).

8 Introduction

To analyze effects of loss-of-function of Lmx1b, part of the Lmx1b locus was deleted (Chen et al. 1998a) (see Materials and Methods section 2.1.1), resulting in a mild phenotype of heterozygous animals and severe defects in homozygous mice. Increasing with age, in Lmx1b +/-, eye defects in the iris were detected while wild-type controls did not reveal any eye alterations (Pressman 2001). Homozygous mutants were characterized by (Chen et al. 1998a; Chen et al. 1998b) and exhibited completely ventralized limbs, lack of the patella and distal ulna, change of hair follicle density, severe skull defects, abnormal sutures between the frontal, parietal and interparietal bones, loss of the cerebellum and the posterior hindbrain, kidney (Morello et al. 2001; Rohr et al. 2002) and ocular defects (Pressman et al. 2000; Pressman 2001). Mice die after birth, presumably as they cannot suckle.

1.1.4 Lmx1b is involved in the Nail-patella syndrome (NPS)

Nail-patella syndrome or onycho-osteo dysplasia (incidence 1:50.000) is a pleiotropic condition exhibiting autosomal-dominant inheritance and was first recognized over 100 years ago (Little 1897). Loss-of-function mutations within the human LMX1B gene, mapped to a 1 cM interval on 9q34 lead to symptoms (Campeau et al. 1995; McIntosh et al. 1997; Dreyer et al. 1998). The syndrome results from heterozygosity and is characterized by dysplasia of the nails and the elbow and hypoplasia of the patellae. NPS is associated with neurological and vasomotor problems (Sweeney et al. 2003), progressive kidney disease (Bennett et al. 1973) and open-angle glaucoma (Beals and Eckhardt 1969; Lichter et al. 1997; Pressman et al. 2000; reviewed by Bongers et al. 2002; Sweeney et al. 2003). Findings in NPS patients resemble the phenotype of homozygous Lmx1b mutant mice (Chen et al. 1998a). The fact that Lmx1b is involved in a human syndrome puts research of the gene on a more fundamental basis and might open future perspectives for the treatment of patients. The first step is a basic knowledge of the expression pattern and an understanding of interactions with other genes. In these fields, this study provides novel information.

9 Introduction

1.1.5 Lmx1b expression is affected in transgenic mice and by overexpression of other limb genes

Two ectodermal factors in the limb are known to interact with Lmx1b expression: Wnt7a, a glycoprotein secreted by the dorsal ectoderm (Parr and McMahon 1995; Riddle et al. 1995) and En1, a homeodomain transcription factor expressed in the ventral ectoderm (Loomis et al. 1996). En1 mutants exhibit a double dorsal limb phenotype and ectopic ventral Lmx1b expression only in the distal limb and expansion of the AER (Loomis et al. 1996). Furthermore, gain-of-function of En1 reduced Lmx1b expression in the dorsal limb mesenchyme by repression of endogenous Wnt7a (Logan et al. 1997).

Targeted disruption of Wnt7a resulted in a ventralized limb, but only the distal part was affected (Parr and McMahon 1995). Early Lmx1b expression was normal in Wnt7a -/- mice and decreased at stages 5/6 in the distal dorsal mesenchyme (Loomis et al. 1998). Ectopic expression of Wnt7a caused induction of Lmx1 in the distal limb but not in the flank (Riddle et al.

1995). Figure I1.2: Expression of Lmx1, Wnt7a and En1 in the wild-type limb (adapted from Johnson and Tabin 1997)

In Wnt7a/En1 double mutants, Lmx1b expression was lost in late limb development only in the distal dorsal limb. No ectopic ventral induction was observed (Cygan et al. 1997). Early Lmx1b expression is induced independently of Wnt7a and En1 function. The phenotype of Lmx1b/Wnt7a and Lmx1b/En1 double mutants is very similar to Lmx1b single mutants. The arrangement of muscles, tendons and bones exhibits a dorsal to ventral conversion (Chen and Johnson 2002).

The Apical Ectodermal Ridge (AER) secretes members of the Fibroblast Growth Factor (FGF) family (Niswander et al. 1993; Fallon et al. 1994; Crossley et al. 1996) and demarcates the ventral border of Lmx1 and Wnt7a expression. Interestingly, AER removal from a HH stage 21 chick limb did not affect Lmx1 expression after 48 hours of reincubation (Vogel et al. 1995).

10 Introduction

1.1.6 Downstream targets of Lmx1b

Lmx1b functions as part of a transcriptional machinery. Still little is known about its regulation and its targets. LDB1, a LIM domain-interacting protein, downregulated LMX1B mediated transactivation (Dreyer et al. 2000) and was shown to interact with Lmx1a (Jurata and Gill 1998; Jurata et al. 1998) independent of the bHLH coactivator E47/shPan1 (German et al. 1992; Johnson et al. 1997; Dreyer et al. 2000). In Lmx1b mutant mice, alpha chains 3 and 4 of type IV collagen were strongly diminished (Morello et al. 2001). Podocin, an integral membrane protein belonging to the stomatin protein family, is encoded by the Nphs2 gene and was completely absent in Lmx1b mutants (Boute et al. 2000; Rohr et al. 2002). In addition, human LMX1B was shown to bind to the putative NPHS2 promotor region and murine Lmx1b bound to a putative enhancer sequence of mouse and human COL4A4, a gene encoding for a3 and a4 chains, indicating a direct regulation of collagen subunits by Lmx1b (Morello et al. 2001; Rohr et al. 2002).

1.1.7 Induction of Lmx1b

1.1.7.1 Wnt7a and FGF8 trigger Lmx1b expression Up to now, no direct upstream genes inducing Lmx1b expression have been determined. Nevertheless, several publications described ectopic induction of Lmx1b. Some interactions were already mentioned in the preceding chapter. In the isthmus organizer located in the mesencephalic/metencephalic boundary, Lmx1b can be induced by FGF8 bead application (Adams et al. 2000). FGF10 was not sufficient to trigger expression of Wnt7a or Lmx1 in the limb bud after foil-barrier insertion lateral to the mesonephric ducts (Ohuchi et al. 1999). In the limb, overexpression of Wnt7a induced strong ectopic expression of Lmx1 in the distal ventral mesenchyme (Kengaku et al. 1998). Ventral proximal limb mesenchyme does not express Lmx1b. Gain- and loss-of-function experiments affected mainly the distal part of the limb (Riddle et al. 1995; Vogel et al. 1995; Loomis et al. 1996; Cygan et al. 1997; Kengaku et al. 1998). In the distal limb, Wnt7a was sufficient to trigger Lmx1b expression. FGF8 induced Lmx1 expression in the brain. It is rational to hypothesize that application of a combination of factors can induce proximal ventral Lmx1b expression.

11 Introduction

To determine the stability and the maintenance of the Lmx1b non-expressing status of ventral proximal cells, a series of grafting experiments and protein applications were carried out.

1.1.7.2 Lmx1b expression in limb culture Culture of HH stage 20-21 chick limbs for 36 hours in the absence of ectoderm leads to loss of expression in the distal dorsal mesenchyme indicating that proximal dorsal mesenchymal cells are capable of the maintenance of Lmx1 expression without continuous inductive signals from the overlying ectoderm (Riddle et al. 1995). The ventral mesoderm in turn did not initiate Lmx1 expression. Limb bud mesenchyme grown on Wnt7a secreting fibroblasts for 36 hours displayed Lmx1 expression in all proximo-distal positions dorsally. In addition, expression was found in the distal ventral mesenchyme but not in the proximal ventral mesenchyme (Riddle et al. 1995). It remained undetermined if other factors are capable of triggering Lmx1 expression in the proximal ventral limb mesenchyme.

1.1.7.3 Sorting out phenomenon Dissociation and reaggregation of limb mesoderm that is inserted into an ectodermal jacket lead to the cancellation of dorso-ventral differences. The dorso-ventral polarity corresponded to the orientation of the ectoderm (MacCabe et al. 1973). A recombinant limb consists of dissociated and reaggregated cells that are inserted into an ectodermal jacket and grafted onto a host. Piedra and colleagues collected cells from the anterior 2/3 of chick wing buds at stage HH 19-21 and examined the recombinant limb generated for expression of different markers (Elisa Piedra et al. 2000). Wnt7a, Fgf8 and En1 expression in the ectoderm were stable. Lmx1 expression was random at timepoint 0, later a downregulation from the central core with a persisting random pattern in the periphery was observed. During outgrowth of the limb, a normal pattern was organized distally forming a sharp transition between randomly distributed cells proximally and the dorsally restricted pattern distally.

12 Introduction

1.2 General limb development

In vertebrates, the tetrapod limbs develop at species dependent somite levels as an outgrowth from the flank, always being located opposite to each other with respect to the midline. Members of WNT and FGF families signaling from the intermediate and lateral plate mesoderm initiate outgrowth of the limb (Searls and Janners 1971; Martin 1998; Kawakami et al. 2001) in chick between stages HH13 and 15 (Hamburger and Hamilton 1951). In mice, forelimbs and hindlimbs appear between E9.0 and E10.0. Two major components form the limb: an ectoderm derived epithelium covers mesenchymal cells of different origin. The largest part of mesenchymal cells giving rise to skeletal elements, cartilage and tendons, are recruited from the lateral plate mesoderm. Structures like muscles, nerves, melanocytes, lymphatics and blood vessel endothelium are derived from extra-limb regions (Hollyday 1983; Serbedzija et al. 1989; Christ and Ordahl 1995; Wilting et al. 1995; Wilting et al. 2000). The limb develops along three major axes, from proximal to distal (shoulder to the finger tips), from dorsal to ventral (knuckles to the palm) and form anterior to posterior (thumb to the little finger). Multiple factors forming the molecular basis for the establishment and maintenance of these axes have been discovered (reviewed by Johnson and Tabin 1997; Niswander 1997; Zeller and Duboule 1997).

Figure I1.3: Chick embryos after 50 and 72 hours of reincubation Induction of forelimb buds in the lateral plate mesoderm occurs between stages HH13 and HH15 in the chick at the level of somites 15 to 20. After 72 hours of reincubation (right scheme), forelimb and hindlimb buds become clearly visible. A-somitic mesoderm; B-lateral plate mesoderm; C-presumptive wing region; FL- forelimb; HL-hindlimb (adapted from Johnson and Tabin 1997)

13 Introduction

Figure I1.4: Scanning electron micrographs of chick embryos incubated for 50 and 72 hours Limbs develop from an outgrowth of the Lateral Plate Mesoderm and are invaded by myogenic precursor cells from the somites. The early limb bud (right photograph) consists of mesenchyme covered by an ectodermal layer. The Apical Ectodermal Ridge (AER) is located at the tip of the limb. (courtesy of Gary C. Schoenwolf, University of Utah School of Medicine; from Johnson and Tabin 1997)

1.2.1 Axis formation

Studies of embryonic limb patterning have provided considerable insight into the general strategies used to establish axial polarity in vertebrate embryos (Johnson et al. 1994). Structures become precisely arranged along the three major axes. Thus, the limb represents a classical model system to study patterning with traditional histological and modern genetic approaches. The molecular basis for anterior-posterior and proximo-distal patterning (Niswander et al. 1993; Riddle et al. 1993; Fallon et al. 1994; Laufer et al. 1994; Niswander et al. 1994) are beginning to be understood. However, mechanisms defining the dorso-ventral axis and detailed function and interaction of Lmx1b remained obscure.

Figure I1.5: Skeletal pattern of the chick wing and the three major axes (modified from Gilbert 2000)

14 Introduction

1.2.2 The proximo-distal axis of the limb

Bones in vertebrates, may it be a bird, a whale, a mammal or a human being are all arranged in a specific system from proximal to distal: The stylopod (e.g. humerus) is connected to the body wall, the zeugopod (e.g. radius-ulna) is located in the middle part, and distally, the autopod consists of structures including carpals and fingers. The Apical Ectodermal Ridge (AER), a thickening in the ectoderm located distally at the interface of the dorso-ventral axis (Saunders 1948; Fallon and Kelley 1977), plays a crucial role in limb bud outgrowth. Removal of the AER leads to truncation of the limb (Summerbell 1974b; Rowe and Fallon 1982) due to decreased proliferative activity in the Progress Zone (PZ). This area ranging within about 200 µm from the tip of the limb, consists of rapidly dividing cells. Grafting of an additional AER into different regions of the limb leads to formation of supernumerary limbs (Saunders 1948; Fallon and Kelley 1977). A new molecular model of limb induction was proposed by Kawakami and coworkers (Kawakami et al. 2001). It was demonstrated that WNT molecules expressed in the somites, in the intermediate mesoderm (IM) and in the lateral plate mesoderm (LPM) act as mediators on the Fgf8/Fgf10 regulatory loop to control limb outgrowth (Lewandoski et al. 2000; Moon and Capecchi 2000; reviewed by Martin 1998). Members of the Fibroblast Growth Factor (FGF) superfamily of secreted factors are capable of directing formation of ectopic limbs (Cohn et al. 1995; Crossley et al. 1996; reviewed by Ohuchi and Noji 1999). Fgf8 is expressed in the AER and interacts with Fgf10 in the Progress Zone triggering distal outgrowth of the limb (reviewed by Cohn and Tickle 1996; Martin 1998). The limbless mutant, an autosomal-recessive mutation, lacks Fgf4, Fgf8, Bmp2, Shh and Msx and displays a complete absence of limbs in homozygote chick embryos (Prahlad et al. 1979; Grieshammer et al. 1996). The dorsal and ventral ectoderm expressed Wnt7a and Lmx1b in the underlying mesoderm. However, in the proximal ventral limb, no ectopic expression was initiated and within the limb, no transcripts for En1 were detected (Grieshammer et al. 1996). This indicates that regulation of Lmx1b in the proximal part of the limb functions in a different way than in the distal part. To analyze these differences was of particular interest in this study.

15 Introduction

1.2.3 The anterior-posterior axis of the limb

The specification of the anterior-posterior axis is established by the Zone of Polarizing Activity (ZPA). This signaling center located at the posterior margin of the limb secretes a diffusible morphogen (Saunders and Gasseling 1968; Wolpert 1969; Tickle et al. 1975). Prior to limb bud formation, information about the anterior-posterior axis is already present (Harrison 1918, 1921; Hamburger 1938). Small blocks of posterior mesenchyme grafted into ectopic anterior regions result in whole mirror-image duplications of the limb (Saunders and Gasseling 1968), depending on the number of transplanted cells (Tickle 1981).

Sonic Hedgehog (Shh) is expressed in the ZPA and was determined to be the molecule responsible for the polarizing activity along the anterior-posterior axis (Echelard et al. 1993; Riddle et al. 1993; Fietz et al. 1994). Wolpert and coworkers proposed a working model in which cells in the ZPA secrete a morphogen forming a gradient with the highest concentration in the Figure I1.6: Expression of Sonic Hedgehog, anterior region. Cell identity is acquired dose- FGFs and Wnt7a in the limb dependent along the anterior-posterior axis (from Johnson and Tabin 1997) (Wolpert 1969). It is still discussed if Sonic Hedgehog acts throughout the limb as suggested from the wide expression pattern of its transmembrane receptor and transcriptional target patched (ptc) (Ingham et al. 1991; Capdevila et al. 1994; Goodrich et al. 1996; Marigo et al. 1996b). Action may be mediated by other Shh dependent molecules like Bone Morphogenetic Proteins (BMPs) (Francis et al. 1994; Laufer et al. 1994; Drossopoulou et al. 2000) and interact with Hox genes (Charite et al. 1994; Yonei et al. 1995; Lu et al. 1997).

1.2.4 The dorso-ventral axis of the limb

Muscles and tendons in vertebrate limbs form a complex array of structures along the dorso-ventral axis. Identification of several molecules involved in dorso-ventral axis formation helped to establish new models explaining development. However, detailed

16 Introduction interactions in particular with molecules expressed proximally of the limb are largely unknown. The current model of dorso-ventral axis establishment is based on classical grafting experiments (MacCabe et al. 1973; Pautou and Kieny 1973; MacCabe et al. 1974; Geduspan and MacCabe 1987, 1989). Reversal of the ectodermal jacket of the wing dorso-ventrally at stage HH14 resulted in wings with mesodermal dorso-ventral polarity. On the contrary, in recombinations at HH16, the ectoderm imposed its dorso-ventral property onto the distal part of the wing (Geduspan and MacCabe 1987, 1989). In Xenopus, rotation of mesenchyme and ectoderm regenerated a normal dorso-ventral pattern including Lmx1 expression at later stages (55) and a reversed arrangement of structures in earlier stages (52) (Matsuda et al. 2001). The ectoderm possesses a signaling capacity imposing its dorsal or ventral status on the underlying mesenchyme (Pautou and Kieny 1973; Geduspan and MacCabe 1986).

Several factors are known to be involved in dorso-ventral axis formation and to interact with Lmx1b (see also section 1.1.5). The dorsal ectoderm expresses the secreted factor radical fringe (r-fng) (Rodriguez-Esteban et al. 1997) and the glycoprotein Wnt7a (Parr and McMahon 1995; Riddle et al. 1995; Vogel et al. 1995) which has induction capacities on Lmx1b located in the dorsal mesenchyme. En1, a homeobox transcription factor is expressed restricted to the ventral ectoderm (Davis and Joyner 1988; Davis et al. 1991; Gardner and Barald 1992) playing a major role in AER positioning together with r-fng (Rodriguez-Esteban et al. 1997). Specific molecules marking the ventral limb mesenchyme have not yet been detected.

The lateral plate mesoderm consists of two sheets, the splanchnopleure and the somatopleure which will give rise to all osteogenic, chondrogenic and fibrogenic cells of the limb (Christ and Ordahl 1995). Michaud and coworkers demonstrated that the ectoderm overlying the medial half of the somatopleure gives rise to the AER. Ectoderm derived from medial locations overlying the somites and the intermediate mesoderm forms the dorsal ectoderm in the limb. Ectoderm covering the lateral plate mesoderm supplies material for the ventral aspect of the limb ectoderm (Michaud et al. 1997). Fate mapping revealed that both, dorsal and ventral ectodermal cells contribute to the formation of the AER (Altabef et al. 1997).

17 Introduction

Unlike the ectoderm, mesenchyme in the mesodermal compartments did not recruit cells from two separate dorsal and ventral lineages (Altabef et al. 1997). Still, tissue recombination experiments changed the polarity of the limb. Flanking of the presumptive limb region with two rows of somites or insertion of a filter laterally to the medial half of the somatopleure resulted in a bidorsal limb. This finding indicated that somites produce a dorsalizing factor and the lateral somatopleure a ventralizing factor (Michaud et al. 1997). Chen and Johnson concluded that the dorsal or ventral aspect of the limb bud ectoderm is determined prior to migration and that dorso-ventral polarity is set by a signaling process from ectoderm to mesoderm (Chen and Johnson 1999). Studies from gain- and loss-of-function experiments of Wnt7a and Lmx1b indicate that the ventral pattern in the limb seems to be the default state (Parr and McMahon 1995; Chen et al. 1998a).

1.2.5 Genetic interactions along the three axes

The signaling systems in each of the three axes do not act independently from each other but integrate in a complex manner. For example, the function of the AER is maintained by signals from the PZ and the ZPA. In turn, the AER retains expression of molecules in the ZPA. Fgf4, expressed in the AER, acts on cells in the PZ and maintains Shh expression in the ZPA (Li and Muneoka 1999). Removal of the AER resulted in decrease of Shh in the posterior mesenchyme. Shh was shown to activate Fgf4 expression in the posterior part of the AER. Signals from the AER and the PZ are codependent and form a positive feedback loop (Todt and Fallon 1987; Laufer et al. 1994; Niswander et al. 1994). To further coordinate proliferation, differentiation, and positional information, the presence of FGFs and Shh is required for the induction of Bmp-2 and HoxD genes (Marigo et al. 1996a; Marigo et al. 1996b). Signaling molecules involved in the formation of structures are not restricted to act within one axis but demonstrate interdependence to coordinate the three dimensional outgrowth of the limb. This seems to be also true for Lmx1b. In a recent review by Chen and Johnson evidence was given for the function of Lmx1b in anterior-posterior patterning (Chen and Johnson 2002). Sonic hedgehog expression in Lmx1b mutants was normal, but loss-of- function of Wnt7a and Wnt7a/Lmx1b exhibited differences in the degree digits and the ulna were affected.

18 Introduction

1.2.6 Establishment of the dorso-ventral boundary and boundaries in general

Boundary formation between fields of gene expression as well as in the separation of tissue structures is an intriguing issue in developmental biology. Two steps are involved: first, the boundary needs to be established. Second, stable maintenance must be achieved even in fields of high proliferation. Vertebrate limbs and Drosophila legs display little structural similarities. For example, the scaffold of limbs consists of a bony endoskeleton while fly legs are covered with chitin (exoskeleton) and do not have bones or related structures. Nevertheless, deuterostomes (chick) and protostomes (Drosophila) show an amazing similarity in the genetic instructions given for limb patterning (Shubin et al. 1997). Shh is expressed in the posterior portion of the vertebrate limb and hedgehog in the posterior part of the Drosophila wing disc. Ectopic anterior expression forms mirror image duplications in both species (Riddle et al. 1993; Basler and Struhl 1994; Chang et al. 1994; Ingham 1994).

Figure I1.7: Expression in the Drosophila wing disc DPP - Decapentaplegic; HH - Hedgehog; EN - Engrailed; WG – Wingless; AP – Apterous; Ser – Serrate; DL – Delta (adapted from Dahmann and Basler 1999)

Important steps in development are balanced between proliferation and differentiation. Furthermore, these steps include segregation and condensation of groups of cells. Still, in most tissues cells can intermingle freely (Lawrence and Morata 1976). Compartments are defined as sets of non-intermingling cells sharing a common cell segregation mechanism divided by boundaries that do not necessarily correspond to morphological landmarks. In Drosophila, the dorso-ventral boundary is formed during the second larval instar. Apterous, the Drosophila orthologue of the murine Lmx1b gene, is restricted to the dorsal compartment of the wing disc and was shown to be required for dorso-ventral

19 Introduction compartmentalization, wing margin formation and specification of the dorsal cell fates (Cohen et al. 1992; Diaz-Benjumea and Cohen 1993; Williams et al. 1993; Blair et al. 1994; Blair 1995). Like in mice, cells in the disc acquire ventral fates if Apterous is deleted (Diaz-Benjumea and Cohen 1993; Williams et al. 1993; Blair et al. 1994). The transcription factor Apterous triggers expression of two Notch ligands, Serrate and Fringe. (Irvine and Wieschaus 1994; Williams et al. 1994; Kim et al. 1995; de Celis et al. 1996). Wingless (Wg), a long-range signaling molecule induced by Notch, is secreted at the dorso-ventral boundary and acts on both compartments (Zecca et al. 1996; Neumann and Cohen 1997), an important fact to position the compartment boundary in the middle of the wing. Lawrence and Struhl presented a multi-step model of Drosophila pattern formation and boundary maintenance (Lawrence and Struhl 1996; reviewed by Dahmann and Basler 1999). First, one group of two separate cell populations starts expressing a ‘selector’ gene, for example Apterous. Then one group secretes a short-range ‘inducer’ to which cells of the other group are sensitive and to which the secreting cells are refractory. Third, cells of the sensitive group respond to the signal and produce a long-range molecule. Proliferation and differentiation of cells is concentration-dependent. In this system, even during massive growth, the boundary can be maintained permanently.

Figure I1.8: Maintenance of a compartment boundary Two founder cell populations are separated from each other. One group of cells starts expressing a ‘selector gene’ (green – ‘on’, white – ‘off’). The state of this selector gene becomes fixed. Establishment of a cell segregation system based on different properties of the ‘on’ and ‘off’ cells maintains a sharp line of division, the compartment boundary. The selector gene induces expression of a short range signaling molecule that acts on ‘off’ cells where expression of a diffusible morphogen (red) is initiated. Patterning of the tissue is dose-dependent and occurs along the diffusion gradient of the morphogen. (adapted from Dahmann and Basler 1999)

20 Introduction

Lmx1b forms a sharp boundary between the dorsal and ventral part, and an additional sharp line of separation towards proximal regions. Limb mesenchymal cells were dispersed and cultured in a micromass and Lmx1b positive cells were stained to determine the sorting out behavior. In addition, Lmx1b expression and in particular the exact location of the boundary was examined at single cell resolution throughout all stages of embryonic development.

1.2.7 Muscle formation

1.2.7.1 Origin of muscle cells and migration of myogenic precursor cells

Figure I1.9: Schematic of myogenic precursor cell migration Myogenic precursor cells (MPC) delaminate from the lateral lip of the dermomyotome and migrate laterally. After invasion of the limb, a dorsal and ventral premuscle mass form. AER, Apical Ectodermal Ridge; MPC, Myogenic Precursor Cells (adapted from Christ and Wachtler 1996)

All muscles of the limb and of the body wall are a derivative of the somites and committed to the myogenic lineage prior to migration (Christ et al. 1974a, b; Chevallier et al. 1977; Christ et al. 1977). The lateral dermomyotomal lips undergo SF/HGF-mediated epithelio-mesenchymal transitions soon after the segregation of the dermomyotome and sclerotome (Bladt et al. 1995; Brand-Saberi et al. 1996b; Heymann et al. 1996). Myogenic muscle precursor cells migrate laterally in the otherwise acellular space between the skin ectoderm and the intermediate mesoderm (Christ and Ordahl 1995). Limb buds develop from somatopleure- derived stationary mesenchyme and are invaded by cells from other locations in a proximo-distal direction. The largest population of these cells is represented by myogenic

21 Introduction precursor cells. These cells replicate for approximately two days at the base of the limb, until they migrate into a dorsal and a ventral zone which form the premuscle masses later on. Embryonic muscle development and control of the muscle size results from a balance between proliferation and differentiation (Patel et al. 2002). Myoblasts are not specified prior to migration concerning the muscle pattern but appear to be determined by the somatopleure-derived mesoderm (Schramm and Solursh 1990; Grim and Wachtler 1991). Muscle differentiation occurs and muscle masses undergo a series of divisions to give rise to individual muscle units which form the mature limb muscles, finally. Formation of muscle fibers is a consequence of fusion of myoblasts (Shellswell and Wolpert 1977; Stockdale 1992; Robson et al. 1994). Reciprocal interactions between muscles and tendons lead to correct patterning and formation of functional units (Kardon 1998). The patterning mechanism responsible for the segregation of dorsal and ventral myogenic cells is not understood at present. The process of somite and limb muscle development has been reviewed recently by (Christ and Ordahl 1995; Brand-Saberi and Christ 1999; Christ and Brand-Saberi 2002; Buckingham et al. 2003).

1.2.7.2 Molecular mechanisms involved in myogenic precursor migration and differentiation Dermomyotomal cells express c-met encoding a transmembrane tyrosine kinase (Cooper et al. 1984), and de-epithelialize under the influence of SF/HGF (Bladt et al. 1995; Brand- Saberi et al. 1996b; Heymann et al. 1996). Loss-of-function of Met or its ligand SF/HGF resulted in lack of muscles in the limb (Bladt et al. 1995; Dietrich et al. 1999), similar to the phenotype of Splotch, a naturally occurring mutation in the Pax3 locus (Franz et al. 1993; Bober et al. 1994). In addition, the hypaxial myogenic lineage depends on other factors originating from the ectoderm and lateral plate mesoderm (Kenny-Mobbs and Thorogood 1987; Pourquie et al. 1993; Fan and Tessier-Lavigne 1994; Cossu et al. 1996; Pourquie et al. 1996; Dietrich et al. 1997). During migration and proliferation, myogenic precursor cells express c-met, Lbx1, Pax3, Pax7, Myf5 and Msx1 (Williams and Ordahl 1994; Jagla et al. 1995; Dietrich et al. 1998; Houzelstein et al. 1999; Kiefer and Hauschka 2001; Swartz et al. 2001). Muscle determining genes remain silent until later stages of development. Myogenic precursor cells and myoblasts are influenced by different common molecules. SF/HGF increases motility and maintains the undifferentiated and proliferative state of

22 Introduction myogenic precursors (Scaal et al. 1999). Application of high concentrations of BMPs, members of the TGF-ß superfamily, are believed to prevent muscle development by induction of apoptosis (Amthor et al. 1998). Members of the FGF family are supposed to promote migration (Webb et al. 1997) and myoblast proliferation while inhibiting differentiation (Edom-Vovard et al. 2001). Premuscle masses can be subdivided into two parts. Proliferating myogenic precursor cells expressing Pax3 and Myf5 are located in the superficial layer, followed by a deep layer of differentiating myoblasts expressing MRFs (Christ and Brand-Saberi 2002). Function of Lmx1b is important for the proper arrangement of dorsal and ventral muscles (Chen et al. 1998a).

1.2.7.2.1 Lbx1 and Pax3 are expressed in myogenic precursor cells In this study, Lbx1 and Pax3 were employed as markers for myogenic precursor cells. The relationship between Lmx1b and Lbx1 expressing cells has never been determined. The homeodomain transcription factor Lbx1, a homologue of the Drosophila ladybird gene is expressed during embryonic development in myogenic precursor cells of the limb, the tongue, the diaphragm and in cells in the CNS and PNS. Lbx1 is downregulated after the onset of differentiation of myoblasts (Jagla et al. 1995; Dietrich et al. 1998; Schafer and Braun 1999). The human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemia (Jagla et al. 1995). The transcription factor Pax3, an orthologue of the Drosophila paired gene, belongs to a group of nine regulatory proteins and is characterized by the presence of homeo- and paired domain motifs (Epstein et al. 1996). Haploinsuffiency of PAX3 in human patients can lead to limb muscle hypoplasia. The chromosomal translocation t(2;13) results in a fusion protein that promotes transcription and was determined to be the cause of pediatric alveolar rhabdomyosarcoma (Barr et al. 1993; Galili et al. 1993; Shapiro et al. 1993; Fredericks et al. 1995). Myogenic precursors of the limb coexpress Pax3 and Lbx1. Splotch mice are devoid of Lbx1 expression. Thus, Pax3 acts upstream of Lbx1 (Mennerich et al. 1998; Dietrich 1999; Gross et al. 2000). Inactivation of Lbx1 did not inhibit delamination of myogenic precursor cells but reduced severely the amount of limb muscles (Schafer and Braun 1999; Gross et

23 Introduction al. 2000). Proper migration to the sites of muscle anlagen failed. All hindlimb muscles and forelimb extensor muscles were absent and the number of forelimb flexor muscles was reduced. Thus, Lbx1 might play a role in guiding migration of myogenic precursor cells and in the maintenance of their migratory potential (Brohmann et al. 2000). In Pax3 mutant mice, cells do not delaminate from the dermomyotome resulting in lack of muscles in the limb (Franz et al. 1993; Tajbakhsh et al. 1997). Myf5 and Pax3 double mutants are devoid of all body muscles (Tajbakhsh et al. 1997). Pax3 is suggested to play a role in keeping myogenic cells in an undifferentiated state as overexpression of Pax3 represses myogenesis (Amthor et al. 1998; Duprez et al. 1998). This is similar to the function derived from in vitro experiments of the homeodomain protein Msx1 (Song et al. 1992; Woloshin et al. 1995). Msx1, one of the three members of the vertebrate Msx homeobox gene family (reviewed by Davidson 1995; Bendall et al. 1999) is expressed in the highly proliferative Progress Zone of the limb, but not in more differentiated cells in the proximal limb (Davidson 1995; Bendall et al. 1999). Hu and coworkers proposed cyclin D1, an inhibitor of differentiation (Walsh and Perlman 1997; Sherr and Roberts 1999) as downstream target of Msx1 (Hu et al. 2001a) which was hypothesized to promote proliferation and inhibit differentiation in different cell lines including myogenic precursor cells (Rao and Kohtz 1995; Skapek et al. 1995; Woloshin et al. 1995; Bendall et al. 1999). In addition, Msx1 reflects proximo-distal differences of differentiation on a molecular level.

1.2.7.2.2 Myogenic regulatory factors After invasion of the limb bud and proliferation, myogenic precursor cells initiate expression of bHLH transcription factors regulating differentiation into muscle. In mice, first expression of Myf5 was detected in the dorso-medial somite at E8.0 and at E9.5 in the lateral hypaxial domain (Ott et al. 1991; Tajbakhsh et al. 1996). Myogenin expression was initiated at E8.5 and MyoD by E9.75. Both genes remain expressed throughout development (Sassoon et al. 1989; Bober et al. 1991; Faerman et al. 1995). Gene targeting revealed the relationship and hierarchy of these genes. MyoD knockout mice are viable and without overt defects in muscles (Rudnicki et al. 1992). Myf5 null mice die at birth due to skeletal defects. Nonetheless, expression of other MRFs and muscle morphology was normal (Braun et al. 1992). In contrast, if both genes, MyoD and Myf5 are deleted, mice lack all muscle fibers and myoblasts.

24 Introduction

Myogenin knockout mice die perinatally as a consequence of a severe deficiency of differentiated muscle fibers (Hasty et al. 1993). In all, results from gene targeting established a model in which MyoD and Myf5 determine the myoblast lineage and act upstream of myogenin and MRF4 which are required for the differentiation and maintenance of the terminally differentiated state (Rudnicki et al. 1993; Megeney and Rudnicki 1995; Rudnicki and Jaenisch 1995). To compare the expression pattern of Lmx1b to muscle markers, antibody staining for MyoD and myogenin was performed.

1.2.7.3 Juvenility hypothesis and proximo-distal development Myogenic precursor cell invade the limb in proximo-distal directions. The Progress Zone is always devoid of myogenic precursor cells (Brand 1985; Brand-Saberi et al. 1989). Specific properties of the extra-cellular matrix are required to increase motility of myogenic cells and to allow movement within the limb. The presence of fibronectin is a prerequisite for migration (Brand-Saberi et al. 1993). The distribution of hyaluronic acid (HA) forms a gradient within the limb (Kosher et al. 1981) coinciding with myoblast localization. HA was shown to enhance movement (Krenn et al. 1991). Migration and formation of premuscle masses depend on homophilic interactions between the myogenic precursor cells and the stationary limb mesenchyme (Brand-Saberi et al. 1996a). Literature notes proximo-distal differences concerning muscle formation. M. Buckingham hypothesized that distal muscles are formed by an earlier migratory population than more proximal muscles (Buckingham et al. 2003) which would be astonishing as myoblast differentiation follows a proximo-distal sequence. At stage HH25, a wave of differentiation starts in the proximal muscle masses of the stylopod and continues distally (Williams and Ordahl 1994). Several dissimilar approaches to investigate in proximo-distal differences revealed remarkable findings. Rotation of the ectoderm along its dorso-ventral axis leads to an inverted pattern of the limb (Pautou and Kieny 1973; MacCabe et al. 1974). The earlier the rotation is performed, the more complete is the inversion of dorso-ventral structures with respect to the proximo- distal axis (Akita et al. 1996).

25 Introduction

Investigation in the regeneration capacity of the limb by implanting additional tissue in different proximo-distal positions showed that the ability of the host tissue declined in more proximal tissue and increased with age (Gay and MacCabe 1984). Distal tendons express the transcription factors Six1, Six2 (Oliver et al. 1995) and the EphA4 receptor (Patel et al. 1996) whereas proximal tendons do not. Thus, the molecular identity of proximal and distal tendons is different. Interestingly, tendon development occurs independent of the presence or absence of muscles (Shellswell 1977; Kieny and Chevallier 1979; Kardon 1998; Brown et al. 1999). Distal fragments of limb buds grafted into the coelomic cavity were invaded by myogenic precursor cells whereas proximal fragments were not (Gumpel-Pinot et al. 1984).

The control of migration of myogenic precursor cells depends on several factors and the changing properties of the environment into which migrating cells enter. Brand-Saberi postulated the so called “juvenility hypothesis” (Brand 1987; Brand-Saberi et al. 1989). The outgrowing limb forms a ‘gradient of juvenility’ (differentiation) along the proximo- distal axis. The state of differentiation, increasing during development, influences directly the capability of myogenic precursor cell migration. Furthermore, migration in interspecific quail-chick grafting experiments was subject to a specific donor-host age relationship. In homotopic transplantations, cells capable of migration invaded the host only if the donor tissue block was older (Brand-Saberi and Krenn 1991).

Manipulations on chick embryos and the generation of quail/chick chimeras contributed a major part to our current understanding of limb development.

The present study concentrates on investigations in properties of cells in particular of the ventral proximal limb bud. Interspecific grafting experiments from proximal ventral locations were carried out, the migration behavior analyzed and compared to results obtained from grafting from other regions. Myogenic precursor cells form a dorsal and ventral premuscle mass but up to now it is unknown if there is exchange between the ventral and the dorsal side marked by Lmx1 expression and if cells underlie a ‘homing effect’. In addition, the stability of proximal Lmx1 expression was of special interest.

26 Introduction

1.3 Mammary gland development

Lmx1b was detected during embryonic and adult mammary gland development which has never been described before in literature. The aim of this study was to examine the expression pattern of Lmx1b during all stages of mammary gland development as whole mounts and by means of histology. In addition, it was assessed if expression is dependent on the functional state of mammary glands or shows variation in the course of development.

Mammary gland development consists of a prenatal and postnatal phase. Unlike other organs, the major part of development occurs after birth. Development is hormone dependent and sexually dimorphic, and consequently different in males and females. The mouse is a representative model for studies on mammary glands, even though significant differences in growth and morphology between rodents and humans exist (Hovey et al. 1999). All information given in the following sections is primarily related to mouse mammary glands. Overall, mammary gland development in mice is a multi-step process and can be divided into four different phases which include the prenatal and postnatal period.

I. Fetal development II. Postnatal development III. Pubertal and postpubertal development IV. Adult development including pregnancy and involution

1.3.1 Fetal development

The mammary gland primordia appear as a downgrowth of the ectoderm between embryonic day 10 and 11 at characteristic locations along the milk line which is a thickened ridge on the ventral part of the embryo (Sakakura 1987). The mammary gland bud becomes lens-shaped by day 12 and bulb-shaped by day 14 and consists of a collection of epithelial cells in the center that are surrounded by condensed mesenchyme. In males, the development is identical until E13 when the testes initiate androgen production (Durnberger and Kratochwil 1980). The epithelial connection between epidermis and the rudimentary gland regresses until E15.

27 Introduction

By E17, a sprout starts invading the mammary fat pad precursor tissue to build up a rudimentary ductal tree with 10 to 15 branching secondary ducts before (Williams and Daniel 1983). Embryonic development was reviewed by Sakakura (Sakakura 1987).

1.3.2 Postnatal development

From birth until puberty which begins at about three weeks of age in mice, the epithelial structures formed before parturition remain quiescent (Daniel and Silberstein 1987; Kratochwil 1987). Only 10-15% of the fat pad are occupied by the parenchyma that has not yet reached the proximal lymph node.

1.3.3 Pubertal and postpubertal development

During the onset of puberty, ovarian steroid levels increase and elongation and bifurcation of mammary ducts occur (Nandi 1959). Elongation of ducts functions by proliferation of cap and body cells in bulbous structures at the tip of ducts named terminal end buds (TEB). (Bresciani 1965; Russo and Russo 1978; Russo and Russo 1980; Silberstein and Daniel 1982; Berger and Daniel 1983). Abundant apoptosis in the body cells forms an inner lumen of the branches (Humphreys et al. 1996; Humphreys 1999). During branching, the connective tissue is broken down to allow extension of ducts (Daniel and Silberstein 1987). Lateral buds either form branches or cleave and give rise to alveolar buds. Massive proliferation continues with peak activity between 3 and 6 weeks of age, until secondary and tertiary ducts finally reach the margins of the fat pad by 3 months of age (Daniel and Silberstein 1987). The TEBs degenerate to blunt ended structures (Humphreys et al. 1996).

1.3.4 Adult development including pregnancy and involution

Mice have 5 pairs of mammary glands, three pairs in the thoracic and two pairs in the inguinal region (Sakakura 1987). In each mammary gland, a lymph node is located proximally which is frequently used as a landmark (Russo and Russo 1996b). The degree of differentiation is higher in the fifth inguinal gland in comparison to the first thoracic gland.

28 Introduction

Initiated by the hormones of pregnancy, a phase of rapid and intense proliferative activity follows which leads to alveolar differentiation. The number of epithelial cells increases by 8 to 12 times (Munford 1963). In the second half of pregnancy termed lobulo-alveolar phase, alveoli cleave further to form milk-secreting units (Cole 1933; Russo and Russo 1978; Russo and Russo 1980). At parturition, alveoli begin abundant milk secretion for the next three weeks. Milk is released to the outside through the primary duct via the nipple. After cessation of pregnancy, a phase of rapid involution decreases the epithelia to adipocyte ratio by apoptosis (Strange et al. 1995). Macrophages and neighboring epithelial cells clear apoptotic cells (Richards and Benson 1971; Fadok 1999). This tissue remodeling process returns the mammary gland in a similar state as before pregnancy and takes about two weeks (Strange et al. 1992).

1.3.5 The cellular composition of mammary glands

Mammary glands consist of two primary components: the parenchyma which forms a system of branching ducts and secretory units and the adipose stroma, a substrate for the development of the ductal system. The stroma contains connective tissue, fibroblasts, unilocular or multilocular adipocytes as well as lymphatics and blood vessels (Neville et al. 1998). The parenchyma can be subdivided into basal luminal epithelial cells and apical myoepithelial cells. A continuous layer of luminal epithelial cells surrounds the central lumen of ducts and expresses different keratins including keratin 18. Myoepithelial cells, positive for smooth muscle actin and keratin 14 rest on the basal membrane that separates the parenchymal and stromal compartments. During lactation, myoepithelial cells act as contractile units to move the milk out of the alveoli and within the ductal system (Richardson 1949; Daams et al. 1987). Myoepithelial cells originate from cap cells of the terminal end bud (Russo and Russo 1996a). After migration into layers closer to the lumen, these cells can give rise to luminal epithelial cells (Williams and Daniel 1983; Daniel and Silberstein 1987). In culture, luminal epithelial cells developed into myoepithelial cells (Pechoux et al. 1999). The basement membrane (BM) is modulated by secretion of basement membrane components like fibronectin, laminin, proteoglycans and type IV collagen by myoepithelial cells (Williams and Daniel 1983; Daniel and Silberstein 1987; Adams and Watt 1993)

29 Introduction

In addition, about 20% of luminal epithelial cells are in direct contact with the BM (Gusterson et al. 1982; Petersen and van Deurs 1988) that is modified by their secretion of proteases (Talhouk et al. 1992).

1.4 Kidney development

The permanent mammalian kidney develops from the metanephros, (detectable from E11 in mice) notably by the rare process of mesenchymal to epithelial transformations (Pritchard-Jones et al. 1990). It is preceded by two transient primitive organs, the pronephros (E8) and the mesonephros (E9.5), all derivatives of the intermediate mesoderm (reviewed by Saxen 1987; Lipschutz 1998). The size, structure, and functional maturity of the mesonephros show a great variation between the species. In human, the mesonephros develops into a functional organ (Martino and Zamboni 1966) while in rodents, the mesonephros remains primitive and non-secretory (Zamboni and Upadhyay 1981; Smith and Mackay 1991). The Wolffian duct, formed initially by fusion of pronephric tubules (Toivonen 1945), sprouts a uteric bud caudally serving as inducer of differentiation of the metanephros (Grobstein 1953, 1955). The nephron is formed by condensations of the metanephric mesenchyme that give rise to the glomerulum, the proximal and distal convoluted tubule including the loop of Henle whereas the stalk of the ureteric bud develops into the ureter, the renal pelvis and the collecting ducts (Saxen 1987; Lipschutz et al. 1996).

1.4.1 WT-1: Wilms’ tumor suppressor gene

Germline mutations of WT-1, a tumor suppressor gene, result in Wilms’ childhood kidney tumor (Haber et al. 1990; Cowell et al. 1991), the most common pediatric renal cancer (Coppes et al. 1993; Huff and Saunders 1993). An amino-terminal regulatory domain and a carboxyl-terminal domain composed of four

Cys2His2 zinc-finger motifs bind to DNA and RNA (Haber and Buckler 1992; Rauscher 1993). Expression of the transcription factor WT-1 was detected first in mesonephric tubules at E9.5 and at later stages in derivatives of the metanephric mesenchyme, the heart mesothelium, the reproductive system and brain including the spinal cord (Pelletier et al. 1991; Armstrong et al. 1993).

30 Introduction

Targeted disruption of WT-1 resulted in complete absence of the metanephric kidney and reduction in the number of mesonephric tubules (Kreidberg et al. 1993). Molecular interactions and results from gene targeting in the kidney were reviewed by Lipschutz (Lipschutz 1998).

The glomerulum consists of different cell types. Only a subset is Lmx1b positive. This study aimed to determine precisely which kind of cells express Lmx1b. WT-1 is known as podocyte-specific marker (Mundel et al. 1997; Pavenstadt 2000) and the distribution of Lmx1b and WT-1 expression are compared.

31 Materials and Methods

2. Materials and Methods 2.1 Animals 2.1.1 Mice

2.1.1.1 Transgenic mice Two different strains of transgenic mice developed by Randy L. Johnson were bred and examined throughout this study. A. Lmx1b 3’LacZ mice were generated to analyze expression of the Lim homeodomain transcription factor Lmx1b in detail. The endogenous Lmx1b gene was not disrupted in this mouse model. LacZ, a reporter gene encoding for ß-galactosidase which can be easily detected by color reaction or immunohistochemistry, was cloned into a HindIII site of the 3’ untranslated region (UTR) of Lmx1b. The complete construct consisted of an internal ribosomal entry site (IRES), the LacZ gene, the phosphoglycerate kinase-1 (PGK) promoter, the neomycin-resistance gene (neo) flanked by LoxP sites (Fig. M2.1).

Figure M2.1: Construct of Lmx1b 3’LacZ (A) and Lmx1b KO mice (B) and the wild-type locus, modified according to Chen et al. 1998a B. Targeted disruption of Lmx1b in mice, referred to as Lmx1b KO, was accomplished by Haixu Chen and Randy L. Johnson (Chen et al. 1998a). Exons 3 to 7 encoding the second LIM domain, the homeodomain and most of the carboxy-terminal region were removed by gene targeting in embryonic stem cells. This resulted in a non-functional Lmx1b protein. Heterozygous animals showed only small defects whereas homozygous mutants die within 24 hours of birth and could not be maintained in a homozygous line.

32 Materials and Methods

2.1.1.2 Establishment and maintenance of the mouse colony Mice were kept in the MD Anderson animal facility under standard conditions. Matings were set up in the evening, females checked at 9 o’clock in the morning for vaginal plugs and noon was considered as embryonic day E0.5 post coitum (dpc). Lines were bred on a mixed B6/129 background. The genotype was determined by Southern blotting and PCR as described in section 2.4.4. Line Strain Line Strain Lmx1b 3’LacZ mixed B6/129 X Wild-type Swiss Webster Lmx1b 3’LacZ mixed B6/129 X Lmx1b KO +/- mixed B6/129

Table 1: Overview of crosses set up during the present study 2.1.2 Chick and quail

Fertilized chicken eggs (Gallus domesticus, strain White Leghorn) and Japanese quail eggs (Coturnix coturnix japonica) were obtained from a local breeder (Bronner in Freiburg- Tiengen, Germany) for interspecific grafting experiments carried out in Freiburg. In situ hybridizations were performed in Houston employing eggs delivered by Charles River (Connecticut, USA). The incubator was set at 38°C and 80% relative humidity. Embryos were staged according to Hamburger and Hamilton (HH) (Hamburger and Hamilton 1951, 1992). Incubation times ranged from 77 hours to obtain HH 18 embryos to 88 hours for HH 22. For grafting experiments, quail eggs were incubated 4 hours earlier than chick eggs which resulted in quail embryos that were one to two HH stages older than the chick hosts.

33 Materials and Methods

2.2 Microsurgery and interspecific grafting experiments 2.2.1 Isolation of mouse embryos

Pregnant mice were killed by cervical dislocation. The peritoneal cavity was accessed, the uterus isolated and embryos dissected in PBS, carefully removing yolk sac and amnion with fine forceps.

2.2.2 Dissection of mammary glands

Mice were killed by cervical dislocation and pinned (ventral side up) on a Styrofoam lid. The skin was cut from the symphysis area towards the presternal region keeping the peritoneum intact. Then the skin was pulled towards lateral until the mammary glands became easily accessible. Lymph nodes and blood vessels were used for orientation. Using stump forceps, tissue located distally of the mammary gland was picked up and removed from the skin cutting with a scalpel. The mammary gland was continuously dissected from distal to proximal and put on a paper of Parafilm (VWR, West Chester, PA, USA) or on a Superfrost Plus Slide (Fisher Scientific, Pittsburgh, PA, USA). Quadriperm dishes (Heraevs Instruments, Inc; Kendro Lab Products, Newtown, CT, USA) were cooled on ice and filled with 4% paraformaldehyde if the mammary glands were destined for antibody staining or with X-gal fixative for X-gal staining. The tissue was transferred immediately into the boxes and fixed for 30 minutes on ice. For X-gal staining and for embedding the standard protocol as described in section 2.5 was used. Mice carry ten mammary glands, five on each side. Preferably, number four (counting from neck to hip, neck is defined as number one) was used for whole mount staining as contamination of this gland with other tissues like fat or muscles was lower compared to the other ones. Mammary glands number two and three were used for sections.

Figure M2.2: Lmx1b 3’LacZ mouse embryo at embryonic stage E15.5 stained with X-gal. Arrows point to mammary gland primordia 1-5. The right hindlimb was removed.

34 Materials and Methods

2.2.3 Quail to chick grafting experiments

Introduction: Quail and chick cells are readily combined as quail cells have specific properties that can be used to distinguish between quail and chick cells by means of histology like Feulgen staining (Le Douarin 1969) or by immunohistochemistry using a species-specific antibody which recognizes quail cells but not chick cells. Hereby it is possible to follow and detect even single quail cells grafted into chick hosts.

2.2.3.1 Materials and surgical instruments

2.2.3.1.1 Tungsten needles Fine instruments were needed to perform microsurgery on the structures of embryos. Tungsten wire at a diameter of 100 µm was cut into pieces of 4 cm and melted into the tip of a Pasteur pipette on a Bunsen burner in a way that the length of the wire outside the pipette was 1.5 cm. Tungsten needles were sharpened electrolytically:

A cathode connected to a transformer set at 8 volts was lowered into saturated NaNO2 in

H2O and the anode linked with the tungsten wire. To complete the circuit, the wire was repeatedly dipped into the solution for several minutes while controlling sharpness under the dissecting microscope. The diameter of the tip was reduced to a few micrometers allowing precise manipulations on the tissue. Before use, needles were washed in boiling water to remove chemicals preventing intoxication of the embryos (Dossel 1958).

2.2.3.1.2 Agarose dishes For grafting experiments, a plate was used to fix embryos and to facilitate dissection of mesenchyme pieces. PBS solution containing 1% agarose was heated up and 6 cm petri dishes were filled by half. A piece of unsharpened tungsten wire was used as a needle to stick the embryo onto the agarose. Agarose dishes for cell culture were made up with sterile ingredients and poured under pathogen-free conditions into petri dishes.

2.2.3.1.3 Dye sticks Parts of embryos were marked using dye sticks to control success of dissection and grafting procedures and to increase contrast. Glass pipettes were melted and drawn out on a Bunsen burner to obtain small round tips. Distilled water containing 2.5% agarose was heated up, 1% nile blue sulfate added and mixed on a stirbar heater. Round ends of the prepared glass pipettes were dipped into the solution for three times.

35 Materials and Methods

2.2.3.1.4 Transfer pipettes Spemann pipettes were used to transfer tissue blocks safely from quail origin into the operation site of chick hosts. A glass tube was melted at one end, pulled long, bent to 30° and the end closed. At 2/3 of the length of the tube, the glass was melted and a bubble blown. The bubble was cracked and the sharp edges smoothened in the fire. The fine tip was cut open and the other end of the tube closed by melting. Finally, a piece of rubber was drawn over the open hole where the bubble was to allow fine regulation of the pressure inside the tube for sucking and blowing out of solution and tissue blocks.

2.2.3.1.5 Other instruments · Leica MZ6 dissecting microscope with cold light supply · A ‘nest’ made of a 6 cm petri dish covered with aluminum foil and softened with several pieces of gauze to house the eggs. · A 5 ml syringe connected to a 20 Gauge needle to suck out yolk and lower the embryo · A metal saw to open the eggs · Pasteur and plastic pipettes to moisten the embryo with Tyrode’s or Locke’s solution · Pieces of paraffin to cover the egg with wax around the operation site · Surgical tape (Durapore-3M) to seal the egg · A pair of forceps and scissors to peel off the shell · Two pairs of fine forceps and scissors for microsurgery · A spoon to transfer embryos · 6 cm petri dishes filled with Locke’s solution or PBS for dissection · An ethanol burner for sterilization of instruments during operations

2.2.3.2 Operations and grafting procedures

2.2.3.2.1 Environment and sterilization Operations were carried out under sterile conditions. Benches and equipment were regularly cleaned with Bacillol and 70% alcohol and exposed to UV – light during the night. Before use, instruments were sterilized at 180°C for two hours and disinfected during operations in the flame of an alcohol burner. To decrease the risk of bacterial infection, penicillin (100.000 I.U./liter) was added to Locke’s solution.

2.2.3.2.2 Preparation of the chick hosts After incubation, chick eggs were put on a light source to determine the position of the embryo which was marked by a cross and put into a nest avoiding unnecessary movements. The egg was wiped with 70% alcohol for disinfection. A hole was stuck into

36 Materials and Methods the pole of the egg where the air bubble was located and 2 ml of yolk removed with a syringe to lower the embryo. Using a metal saw, a square of 1 cm x 1 cm was cut into the shell and the piece carefully removed with forceps. A drop of Locke’s solution or Tyrode’s solution (Sigma-Aldrich Corp., St. Louis, MO, USA) was put on top of the egg shell membrane before opening the egg completely. Some more drops were added on the embryo to ensure moistening. The embryo was staged according to Hamburger and Hamilton (1951), the result written on the shell and the egg sealed with surgical tape and put back into the incubator until the quail donor was prepared. This procedure avoided unnecessary cooling down of the egg and prevented an operation lag that would have affected in particular short reincubation times. Furthermore, as not all embryos were at the same stage but several eggs were staged and reincubated as described, the perfectly fitting host could be chosen for grafting later on thus taking care of the correct donor-host age relationship.

2.2.3.2.3 Preparation of quail donor mesenchyme Quail eggs were put on a nest, a needle inserted at the pole where the air bubble was located and 1.5 ml yolk sucked out with a syringe. The shell was cut open and after detecting the embryo, the egg was opened completely and the embryo cut out with ophthalmologic scissors and transferred with a spoon into a 6 cm petri dish containing Locke’s solution. Staging was performed according to Hamburger and Hamilton as well. Yolk sac, amnion, head and heart were dissected from the embryo and then it was transferred on an agarose plate filled with Locke’s solution, positioned and fixed with a piece of tungsten wire. Dye sticks helped to stain the ectoderm before its removal with sharpened tungsten needles.

2.2.3.2.4 Grafting procedure in general Prepared like this, the quail embryo was put aside while a suitable chick host, two HH – stages younger than the quail donor, was chosen and accessed in the egg. Locke’s solution was added on the chick embryo to raise it over the level of the shell which was possible due to the surface tension of the water and helped to put the embryo in good light and made it easily accessible for microsurgical manipulation. The vitelline membrane and the amnion were opened over the area of the forelimbs using fine forceps. The graft was dissected from the quail donor, as described in detail in the operation section, visualized with nile blue sulfate and transferred into the chick host egg using

37 Materials and Methods pipettes described above. Sharpened tungsten needles and ophthalmologic instruments served to incise the host limb and to position the graft. After the manipulation was completed, 2 ml of yolk were removed from the egg with a syringe to lower the embryo. It was double checked if the stained graft was still in good position after lowering and moistening of the chick host. If no change had occurred, the egg was sealed with surgical tape, the operation number and time marked on the shell and put back into the incubator. Notes were taken for each operation concerning time, stage, exact position and size of the graft. After the desired reincubation time, eggs were opened, embryos checked and notes made about the graft. Embryos were dissected out of the egg, transferred into a petri dish containing PBS, dissected, washed and fixed in 4% paraformaldehyde over night.

2.2.3.2.5 Schemes of grafting procedures

Operation From quail To chick OP Reincubation Fixation stage stage OP A ventral dorsal proximal 21-22 8-22, 48 h 26-28 OP B dorsal proximal ventral proximal 21-22 48 h 26-28 OP C ventral proximal homotopic 21-22 48 h 26-28 OP D dorsal proximal homotopic 21-22 48 h 26-28 OP E ventral proximal Progress Zone 20-21 48 h 26-28 OP F ventral proximal central 21-22 48 h 26-28

Table 2: Overview of interspecific grafting procedures

Different transplantations were carried out (see schemes) excising a block of quail mesenchyme from various positions of the wing and inserting it into a younger chick host. Mesenchyme blocks measuring about 200 µm from one side only (dorsal OR ventral) were dissected out and grafted in the following ways:

38 Materials and Methods

2.2.3.2.5.1 OP A Lmx1b-negative wing mesenchyme from the ventral side and from different proximo-distal positions of a two stages older quail donor was grafted into the dorsal Lmx1b-positive part of a chick host wing at HH stages 21-22.

Figure M2.3: OP A - ventral to dorsal

2.2.3.2.5.2 OP B Wing mesenchyme from the Lmx1b-positive dorsal side and from different proximo-distal positions of a two stages older quail donor was grafted into the ventral part of a chick host wing at HH stages 21- 22.

Figure M2.4: OP B - dorsal to ventral

2.2.3.2.5.3 OP C Wing mesenchyme from the ventral side, mainly from proximal posterior positions of a two stages older quail donor was grafted homotopically into the wing of a chick host at HH stages 21-22.

Figure M2.5: OP C - ventral to ventral

2.2.3.2.5.4 OP D Wing mesenchyme from the dorsal side and from different proximo-distal positions of a two stages older quail donor was grafted homotopically into the wing of a chick host at HH stages 21-22.

Figure M2.6: OP D - dorsal to dorsal

39 Materials and Methods

2.2.3.2.5.5 OP E Lmx1b-negative wing mesenchyme from the ventral side of a two stages older quail donor was grafted into the Progress Zone underneath the AER of a chick host at HH stages 21-22. The graft was located between dorsal and ventral distal mesenchyme. The same operation was performed using LacZ-positive mesenchyme from Lmx1b 3’LacZ transgenic mice at embryonic stage E11.5 which was grafted into the Progress Zone of a host Figure M2.7: OP E - ventral into the limb of a Swiss Webster wild-type mouse embryo Progress Zone of the same stage.

2.2.3.2.5.6 OP F Wing mesenchyme containing migratory cells from proximal positions in the ventral side of a two stages older quail donor was grafted centrally with respect to all three axes into the wing of a chick host at HH stages 21-22. The graft was positioned in the future chondrogenic zone between the dorsal and the ventral side. Figure M2.8: OP F - ventral to central

2.2.3.2.6 Reincubation and fixation Embryos were reincubated for 48 hours, removed from the egg and dissected in PBS. Notes were taken on HH stage, reincubation time, morphology of the operated wing. The manipulated wing was compared to the contralateral one. Then embryos were fixed in 4% paraformaldehyde over night and processed for whole mount or histological analysis.

40 Materials and Methods

2.3 Cell culture and in vitro culture of embryos Throughout this study, different cell culture methods were employed to analyze migration behavior of a defined cell mass and to study the effects of protein application on mouse embryos which could be achieved only in an in vitro system.

2.3.1 Cell culture

All cell culture experiments were performed in a sterile environment. The temperature of the incubator was set to 37°C and the air contained 5% CO2. Protocols for cell culture were adapted from Ian Freshney (Freshney 1998). The following procedures were applied to 3T3 cells while micromass culture will be described in section 2.3.2.

2.3.1.1 Media and solutions

2.3.1.1.1 Media A for cell culture Complete serum containing medium consisted of 90% Dulbecco’s Modified Eagle Media (myogenic-1X, high glucose, L-glutamine, pyridoxine hydrochloride, no sodium pyruvate) from Gibco BRL, Life Technologies Inc. (Rockville, MD, USA), 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin from Gibco BRL. 50 µg/ml ascorbic acid was added into media for micromass cultures but not for 3T3 cells.

2.3.1.1.2 Media B for in vitro culture of embryos Complete medium for culturing embryos or parts of embryos contained serum-free, defined BGJb medium from Gibco BRL supplemented with 0.2 mg/ml ascorbic acid, 50 U/ml penicillin and 50 µg/ml streptomycin.

2.3.1.2 Recovery of cells Tubes were thawed in a waterbath at 37°C and the content transferred into a flask. Media was added slowly to prevent osmotic damage of the cells. After transfer into a 15 ml tube, cells were spun down in a clinical centrifuge at 3500 rpm for 3 minutes. The supernatant was discarded, fresh media added and the washing procedure repeated to eliminate DMSO. Then cells were resuspended in media, split 1:10 and incubated in 10 cm petri dishes.

2.3.1.3 Splitting of cells The density of the cell culture and the morphology of the cells were controlled every day. When cells became close to confluent, splitting was performed to allow continued growth under appropriate conditions. For this procedure, media was removed and cells washed

41 Materials and Methods with 12 ml sterile PBS. 3.5 ml trypsin were added and the dishes incubated for 3 to 5 minutes at 37°C to remove cells from the plate. Further enzymatic reaction was inhibited by addition of media and cells were broken up completely with a plastic pipettor and transferred into a 15 ml tube. Centrifugation at 3500 rpm for 3 minutes pelleted the cells. The supernatant was removed, cells resuspended in 10 ml media and split 1:5 or 1:10 into new petri dishes.

2.3.1.4 Long term storage of cells When cells had become 60 to 70% confluent, media was removed, cells washed with 12 ml of sterile PBS and broken up with 3.5 ml trypsin (0.25%, 1 mM EDTA) during a 3 to 5 minute incubation time at 37°C. 7.5 ml of media were added, the cells broken up completely with a pipettor, transferred into a 15 ml tube and pelleted. The supernatant was removed and the cells resuspended in 3 ml freezing media (155 ml DMEM, 20 ml fetal calf serum, 50 U/ml penicillin and 50 µg/ml streptomycin and 24 ml DMSO). Aliquoted into cryogenic vials, cells were frozen slowly protected by Styrofoam plates in the -80°C freezer. The next day, vials were transferred into a liquid nitrogen storage tank. When the cells were needed again, recovery was performed as described in 2.3.1.2.

2.3.1.5 Culture in a “hanging drop” This variation of the culture technique was used to obtain a dense culture of protein secreting cells that could be cut into small pieces and grafted into host embryos. Cells were cultured until 80-100% confluent and then processed as described above for the splitting of cells. The pellet was resuspended in 0.5 ml to 1 ml of media, put in small drops on a 100 mm petri dish. After incubation for 2 hours when cells had attached to the plate, the dish was turned by 180°, the lid moistened with media and put back into the incubator or used for the experiment immediately. Alternatively, after incubation of the drops for 2 hours, media was added, similarly to the micromass culture procedure and reincubated for several hours to one day. The cells formed a quite solid tissue dot that could be easily dissected into smaller portions for grafting.

2.3.1.6 Trypan blue staining For viability assessment of cells, a 1% trypan blue solution was mixed 1:1 with an aliquot from a cell suspension and incubated for 5 minutes at room temperature and evaluated under the microscope. Dead cells took up the trypan blue whereas life cells staid unstained.

42 Materials and Methods

2.3.1.7 Coating of coverslips Coverslips needed to be precoated before use, as otherwise cells did not attach properly. Two methods were used:

2.3.1.7.1 Coating with Poly-D-Lysine Coverslips (Fisher Scientific) were washed twice in water and cleaned in nitric acid for two days. After rinsing four times in water, coverslips were autoclaved and stored in a sealed petri dish. If precleaned coverslips were used, these initial washing steps were omitted. Coverslips were put into sterile 6-well-plates and 1 ml of a 100 µg/ml Poly-D-Lysine stock solution from Sigma were added and put on a slow rotator over night. Then coverslips were washed three times with filtered and autoclaved water, dried in an oven at 50°C for several hours and stored at 4°C in a box sealed with parafilm until use.

2.3.1.7.2 Coating with gelatin A second method employed to make slides more sticky which was less effort and proved to be as efficient for the cells used here. Coverslips were emerged in 0.1% gelatin solution in sterile H2O and put on a rotator over night. After washing, coverslips were dried and stored sterile until use.

2.3.1.8 Heparin acrylic beads soaked in recombinant protein Glass pipettes were drawn on a gas burner in a way that mainly small beads would enter the glass tube. A small amount of beads was sucked into the pipette from the stock of heparin acrylic beads (Sigma) and transferred into 6 cm petri dishes. The beads were washed once and soaked over night in D-PBS at 4°C. Recombinant mouse FGF-8b from R&D Systems (Minneapolis, MN, USA) was thawed at room temperature. Addition of 25 µl 0.1% BSA fraction V in PBS resulted in a final concentration of 1 mg/ml FGF-8b. After careful trituration, the tube was spun for 30 seconds at 4000 rpm. Beads of the same size and round shape were washed twice in sterile PBS and transferred into a new petri dish where excess PBS was removed. 12 µl of the reconstituted FGF-8b were added. The margin of the petri dish was moistened with several drops of D-PBS to prevent evaporation (and thereby changing the concentration) of the FGF-8b solution. The petri dish was sealed with parafilm and stored at 4°C until use.

43 Materials and Methods

2.3.2 Micromass culture

Introduction: The micromass culture technique consists of high density dot cultures of dissociated limb bud cells (Ahrens et al. 1977). One advantage of this method is that small numbers of cells were sufficient to obtain many three-dimensional and multilayered cultures which were an important reason to make use of this method in this work. Originally this technique was developed to study chondrogenesis but it has been extended to several other systems. In vitro recapitulation of pattern formation, morphogenesis and differentiation can be studied. The following protocol has been adapted from K. Daniels (Bronner-Fraser 1996)

2.3.2.1 Isolation of limb mesenchyme cells Two rows of 1.5 ml tubes were prepared and labeled with numbers. One row was filled with X-gal for staining of the heads of the embryos to check if embryos were positive for LacZ. The other row was filled with D-PBS where the thorax including the limbs was inserted later on. Embryos were collected from crosses of Lmx1b 3’LacZ transgenic mice, dissected in PBS and transferred into sterile PBS to reduce blood cell contamination. Heads were put into X- gal and incubated at 37°C. This step was omitted when embryos from matings were used that should give only heterozygous or homozygous progeny according to Mendelian rules. Limbs and thorax were put into corresponding tubes in D-PBS and stored on ice until the color reaction could be seen hereby determining the genotype of the embryos. Hindlimbs and forelimbs were used in the same assay to obtain a sufficient amount of limb mesenchyme cells. Casein is the substrate of dispase, an enzyme produced by Bacillus polymyxa that is inhibited by EDTA but not by serum (Griffin and Fogarty 1971; Fogarty and Griffin 1972). Dispase has proven to be a rapid, effective but gentle agent for separating intact epithelial sheets (Stenn et al. 1989). Dispase solution was sterilized by filtration through a 0.22 µm filter membrane and employed in this experiment to remove the ectoderm preserving viability of the cells. Only LacZ positive limbs were utilized, the D-PBS was replaced by prewarmed calcium- free and magnesium-free D-PBS containing 0.2% dispase II (1.21 U/mg) from Gibco BRL and incubated for half an hour at 37°C in a Vortemp shaking incubator from Labnet Inc. (Edison, NJ, USA) rotating slowly. Then tubes were inverted gently and the ectoderm came off.

44 Materials and Methods

If necessary, leftovers of ectoderm were dissected off manually with the help of tungsten needles under the dissecting microscope and limbs were separated from the trunks. The limb bud mesenchyme collected this way was transferred into a 1.5 ml tube containing D- PBS, triturated carefully, 0.2% dispase added and incubated for 10 minutes at 37°C. Cells were triturated again, spun down in a microfuge at 1000 rpm for 2 minutes and redissolved in PBS or media to eliminate dispase in the suspension.

2.3.2.2 Single cell suspension and determination of the cell concentration The sorting out phenomenon could only be studied if it was ascertained that at the very beginning of the culture no complexes like undissociated cells were present and all changes in morphology of the dot culture were due to migration and differentiation behavior of the cells. Therefore cells were filtered to obtain a single cell suspension and the cell number was adjusted to the standard of micromass culture: 2 x 107 cells/ml.

The filtration apparatus was self-assembled and proved to be very effective. A round piece of nylon membrane with a pore size of 20 µm from Spectrum Laboratories (Rancho Dominguez, CA, USA) was fitted onto the bottom 5 mm of a blue tip that had been cut off and both were inserted carefully into a 1.5 ml Eppendorf tube ensuring that the nylon membrane covered the bottom of the blue tip completely and left no spaces.

20 µl of media were added into the tube, then a Figure M2.9: Filtration apparatus maximum of 500 µl of cell suspension into the top part.

After centrifugation at 1000 rpm for 60 seconds, more suspension was added and centrifuged again. The tube was opened and a 20 G needle connected to a syringe poked into the bottom of the 1.5 ml tube to transfer the cells into a new tube. An aliquot was checked under the microscope for viability assessment with trypan blue and to ensure the success of the filtration procedure.

Next, the number of cells was to be determined with a hemocytometer slide from Neubauer. Consisting of a chamber of defined size 0.1 mm deep and a central grid of 1 mm2, the volume could be calculated (10-4 ml). The distance between the thick lines of the grid was 200 µm and between the small lines 50 µm. Before applying 10-15 µl of a

45 Materials and Methods suspension into the hemocytometer, cells were triturated briefly to obtain a homogeneous distribution. Cells in the small or big squares were counted depending on the number of cells, added together and the concentration of the cell suspension was calculated. By adding more media or centrifuging cells and removing supernatant, the number of cells for micromass culture experiments was adjusted to 2 x 107 cells/ml.

2.3.2.3 Culture 10 µl for each culture of a 2 x 107 cells/ml suspension were put as a single drop without bubbles on precleaned and precoated coverslips or slides housed inside petri dishes. A circle of media was spread around the glass carriers to humidify the chamber. Dots were checked under the microscope for equal distribution of the cells. During incubation for 2 hours at 37°C and 5% CO2, cells attached to the coverslips or slides and then dishes were flooded gently with prewarmed complete media to ensure sufficient nutrition. Reincubation times ranged from 2, 12, 24, 36 to 72 hours. Media was exchanged every day.

2.3.2.4 Fixation and color reaction Media was removed and cells washed carefully in PBS. X-gal fixative was applied for 4 minutes for X-gal staining, then cells were washed in PBS, the pH adjusted in equilibration solution for 10 minutes and then stained in filtered X-gal for 24 hours at 37°C. The culture was washed in PBS, dehydrated in ethanol series and mounted. For all other purposes, cells were fixed in 4% paraformaldehyde for 10 minutes, counterstained and mounted.

2.3.2.5 Alcian blue staining Micromass cultures were fixed in 4% paraformaldehyde for two hours at room temperature and washed in PBS. Alcian blue staining solution consisting of 1% Alcian blue 8GX in 0.1 N hydrochloric acid was applied for 30 minutes. After dehydration in 95%, 100% and 100% for 30 seconds each and in histoclear for 10 minutes, slides were mounted in Permount. After staining, the distribution of the cells was analyzed and compared to cultures from the same origin fixed at other timepoints.

46 Materials and Methods

2.3.3 Whole embryo culture

2.3.3.1 Preparation of the embryos Embryos were prepared and genotyped as described in the micromass culture section. The head and the abdominal region between fore- and hindlimbs were dissected off. Thus, dorsal and ventral could be easily distinguished. The limb level was kept in D-PBS at 4°C until the color reaction showed the genotype of the heads. For ectoderm removal, the embryo parts were incubated in prewarmed D-PBS containing 0.2% dispase II for 30 minutes at 37°C in a Vortemp shaking incubator rotating slowly. If necessary, the ectoderm was peeled off mechanically with tungsten needles under the dissecting microscope. After washing in complete media, the mesenchyme parts were transferred onto a filter membrane of 12 µm pore size inside a Costar Transwell dish no. 3403 from Corning Inc. (Corning, NY, USA), floated on complete media B and incubated for 24 hours. Development was only slightly delayed in comparison to in vivo outgrowth (Zuniga et al. 1999).

2.3.3.2 Manipulations on cultured embryos Two series of experiments on cultured mouse embryos were performed. In set A, a block of ventral proximal limb buds mesenchyme was collected from Lmx1b 3’LacZ transgenic mouse embryos at embryonic stage E11.5 and grafted into the Progress Zone of a limb bud of a wild-type Swiss Webster mouse embryo at the same stage. See grafting scheme M2.7 – OP E. Limbs were treated and cultured for 24 hours as described above, fixed, stained with X-gal and analyzed.

In set B, limb bud mesenchyme devoid of ectoderm after dispase II treatment and derived from Lmx1b 3’LacZ transgenic mouse embryos was exposed to different proteins produced by 3T3 cells and from heparin acrylic beads, cultured for 26 hours and processed for X-gal staining. In different assays, the following combinations were applied to Lmx1b-negative ventral proximal limb bud mesenchyme: 1. 3T3 cells secreting Wnt7a and FGF8b beads; 2. FGF8b beads only; 3. 3T3 cells secreting Wnt7a

3T3 cells obtained from Chen-Ming Fan (Baltimore, MD, USA) were cultured as described in 2.3.1. In order to obtain a cell mass that could be grafted, cells were cultured in a ‘hanging drop’ as described in 2.3.1.5 before use and transferred onto the mouse limb

47 Materials and Methods mesenchyme. As control, 3T3-Wnt7a cells were cultured separately in the same assay as the limb buds and stained with X-gal. No color reaction was detected. Preparation of heparin acrylic beads soaked in FGF8b was performed as noted in section 2.3.1.8.

2.3.3.3 Fixation and staining Cultured embryos were fixed in X-gal fixative on ice for 15 to 20 minutes, rinsed twice in equilibration solution for 10 minutes and stained with X-gal at room temperature. The tissue was postfixed and stored in 4% paraformaldehyde. The same solutions were used as for whole mount X-gal staining in section 2.5.1.1.

48 Materials and Methods

2.4 Molecular biology 2.4.1 General methods

2.4.1.1 Gel electrophoresis Introduction: DNA and RNA fragments of 100 base pairs to 20 kbps can be separated by running on an agarose gel. Negatively charged at neutral pH, nucleotides move towards the positive electrode and can be visualized under ultraviolet light by adding ethidium bromide, a fluorescent dye that intercalates between base pairs. If not noted otherwise, 1% agarose was dissolved in TAE buffer containing 3 µl/100 ml from a 10 mg/ml ethidium bromide stock solution. 10% loading buffer (25% bromophenol blue, 25% xylene cyanole FF, 30% glycerol, water) was added to the sample. In each assay, a marker of known length (1 kb DNA ladder) was run additionally to determine the size of the fragments. Photographs were taken under ultraviolet light.

2.4.1.2 DNA extraction from agarose gels DNA was extracted from agarose gels using the Qiagen gel extraction kit (Qiagen Inc., Valencia, CA, USA) according to the instructions of the Manufacturer.

2.4.1.3 LB/ampicillin plates 20 g/l LB broth (10 g/l digest of casein, 5 g/l yeast extract, 5 g/l sodium chloride) and 15 g/l Bacto-Agar were dissolved in water and autoclaved for 20 minutes. When cooled down to 50°C, ampicillin was added to a final concentration of 100 µg/ml and poured into sterile petri dishes.

2.4.1.4 Transformation of bacteria JML09 competent cells from the house stock were thawed slowly on ice. 4 µl of the DNA obtained from a filter paper was added to 100 µl of thawed competent cells and incubated on ice for 30 minutes. Cells were heat shocked for 45 seconds at 42°C, cooled on ice for 2 minutes and then 400 µl of antibiotic free TB media were added. After incubation at 37°C for one hour shaking, 100 µl were spread evenly on a LB/ampicillin plate and the remaining bacteria centrifuged briefly at 14.000 rpm. All but 100 µl of the supernatant was discarded, the bacteria resuspended and plated out on a second LB/ampicillin plate. Incubation was performed over night at 37°C.

49 Materials and Methods

2.4.2 Large plasmid DNA preparation

The protocol for the large plasmid preparation was derived from the Molecular Cloning book by Sambrook (Sambrook et al. 1989).

2.4.2.1 Amplification and extraction 500 ml TB media containing 100 µg/ml ampicillin were inoculated with a single colony of bacteria and incubated vigorously shaking at 37°C over night. Cells were transferred into 250 ml centrifuge bottles and pelleted in a Sorvall Instruments centrifuge (Du Pont, USA) equipped with a JA-14 rotor at 5000 rpm for 10 minutes. The supernatant was discarded and cells resuspended in 10 ml of solution I (50 mM Glucose, 25 mM Tris-HCl pH 8, 10 mM EDTA pH 8). Cells were split up by adding 20 ml of 10 N sodium hydroxide, 20% SDS solution and incubated for 5 minutes at room temperature. The mixture was neutralized with 15 ml ice-cold solution II (3 M potassium acetate, 5 M glacial acetic acid) and stored on ice for 5 minutes. After centrifugation at 6000 rpm for 5 minutes, the solution was filtered through cheesecloth into a new centrifugation bottle.

2.4.2.2 Precipitation and purification For precipitation of the DNA, 90 ml ethanol were added and the tube incubated at room temperature for 10 minutes. Nucleic acids were pelleted at 7000 rpm for 10 minutes and the supernatant was discarded. 100 ml of 70% ethanol were added, the pellet dislodged and centrifuged at 7000 rpm for 10 minutes. The supernatant was discarded and the pellet dried in an incubator set at 37°C for 10 minutes. The pellet was resuspended in 3.25 ml TE-8 and 3 ml were transferred into a 12 ml centrifuge tube. Purification of the plasmid DNA was performed over a cesium chloride gradient by adding 3.5 g cesium chloride and 0.2 ml of a 10 mg/ml ethidium bromide solution. After centrifugation at 6000 rpm in a JA-17 rotor for 5 minutes, the supernatant was transferred into a 3.9 ml quick seal tube sealed by melting. Strong centrifugation at 100.000 rpm in an ultracentrifuge from Beckman Instruments (Fullerton, CA, USA) overnight separated the different contents: Purified plasmid DNA with intercalated ethidium bromide was located in the intermediate layer. On top of it a second layer containing bacterial DNA was visible. A hole was pocked into the tube underneath the intermediate layer and the plasmid DNA removed with a syringe and transferred into a 1.5 ml tube. An equal volume of isoamyl alcohol was added to remove the ethidium bromide from the DNA. After one minute of centrifugation at 14000 rpm, the upper layer was discarded and

50 Materials and Methods the procedure repeated. Then the solution was split into several tubes, 3 volumes of 70% ethanol were added and put on dry ice for 10 minutes. As soon as the CsCl had redissolved, the DNA was pelleted in a microfuge at 4°C for 10 minutes. The supernatant was discarded and the DNA washed again in 1 ml of 70 % ethanol, centrifuged at room temperature for 5 minutes, the supernatant discarded and the pellet air dried. 300 µl TE-8 were added to dissolve the DNA.

2.4.2.3 Spectroscopy The DNA concentration and the purity of samples were determined by spectral photometry. The optic density (OD) defined as the amount of substance dissolved in 1 ml giving an absorbance reading of 1 in a chamber of 1 cm path length, was measured at 260 nm (peak of nucleic acids), 280 nm (peak of proteins) and 320 nm (dirt and contamination). Absorbance at 260 nm of 1 corresponds to 50 µg double stranded DNA.

The A260/A280 ratio of pure DNA is 1.9 and should be greater than 1.8. A value lower than 1.9 indicates protein contamination. The OD at 320 nm was used for absorbance correction.

2.4.3 Preparation of digoxygenin labeled probes

2.4.3.1 Linearization of plasmids 5 µg of DNA that had been cloned into a polylinker site of an appropriate transcription vector containing promoters for SP6, T7 or T3 polymerases, were cut with the appropriate restriction enzyme in an assay containing: DNA, restriction enzyme, a suitable buffer and

H2ODEPC. After incubation for 2 hours at 37°C, linearization was checked on a 1% TBE gel.

chick Lbx chick Lbx chick Lmx1 chick Lmx1 chick Pax3 SP6 sense T7 AS T3 sense T7 AS AS H2Odepc (in µl) 83 83 81 81 87 Buffer (10x) Buffer B Buffer 3 Buffer L React4 Buffer 3 Buffer (in µl) 10 10 10 10 10 DNA (plasmid, 5µg) 5 5 7 7 1 Manufacturer Boehringer Biolabs Boehringer Biolabs Biolabs Enzyme (10 U) EcoRV BamHI KpnI SpeI BamHI Enzyme (in µl) 2 2 2 2 2 100 µl 100 µl 100 µl 100 µl 100 µl Table 3: Assays for linearization of Lbx, Lmx1 and Pax3

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2.4.3.2 Purification and precipitation of DNA To free DNA from protein contamination, phenol/chloroform (p/c) extraction was performed. 100 µl of DNA from linearization, 100 µl TE-8 buffer, 200 µl phenol/chloroform were mixed, centrifuged and the supernatant taken out. TE 8 was added, the tube vortexed and centrifuged and the supernatant saved again. DNA was precipitated by adding 1/10 of the volume of 3 M sodium acetate pH 5.2 and 2.5 volumes of ice cold ethanol, frozen at -80°C and then centrifuged for 30 minutes. The supernatant was discarded, 70% ethanol added, mixed and centrifuged again. The supernatant was removed, the DNA air dried and redissolved in TE-8.

2.4.3.3 In vitro transcription - labeling with digoxygenin RNA labeling with digoxygenin-UTP by in vitro transcription was performed according to the Manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany). Boehringer Promega

H2Odd, RNase free 10.5 µl 6.5 µl Transcription buffer 2 (10x) µl 4 (5x) µl DTT 0.1 M 0 2 µl DIG RNA labeling mix, 10x 2 µl 2 µl Linearized plasmid DNA (1µg) 4 µl 4 µl RNase- Inhibitor 0.5 µl 0.5 µl RNA polymerase (SP6, T7, T3) 1 µl 1 µl 20 µl 20 µl

Table 4: In vitro transciption assay

After 2 hours of incubation at 37°C, unincorporated nucleotides were removed from the RNA probe either by using the QIAquick PCR purification kit protocol (Qiagen Inc., Valencia, CA, USA) or by ethanol precipitation with addition of glycogen and lithium chloride. The probe was redissolved in 50 µl TE-8 and 50 µl hybridization buffer and stored at -20°C.

2.4.4 Genotyping of transgenic mice by Southern blotting and PCR

2.4.4.1 Southern blotting Introduction: Southern blotting, named after its originator Edwin Southern in 1976, is a technique employed for the detection of specific DNA sequences. After digestion with restriction enzymes, gel electrophoresis separates the fragments. The DNA is transferred onto a membrane and a radioactive labeled probe is used for hybridization to the fragment of interest. Autoradiography reveals the position of the DNA (Lodish et al. 2001).

52 Materials and Methods

2.4.4.1.1 Agarose gel extraction In order to obtain a probe for Southern blot analysis, fragments cloned into Bluescript vectors were extracted from the constructs by digestion with specific restriction enzymes (see table), and run on a 1.25% agarose gel containing ethidium bromide. Fragments of interest were cut out and DNA was extracted and purified using the Qiagen gel extraction kit (Qiagen Inc., Valencia, CA, USA) following the recommendations of the Manufacturer and the amount of DNA quantified. mouse construct made by restriction enzyme fragment of interest Lmx1b 3’ LacZ 47BI Bgl R RL Johnson BglII and NcoI 0.7 kb Lmx1b KO -/- 47B2 RL Johnson Xba 0.9 kb Table 5: Probes for Southern blotting

2.4.4.1.2 Tail DNA extraction and digestion Mice were individualized by ear clipping and DNA collected from the distal tip of their tails. 700 ml lysis buffer (50 mM Tris pH 7.5, 50 mM EDTA pH 8.0, 100 mM sodium chloride, 5 mM DTT, 0.5 mM spermidine-HCl, 1% SDS) and 40 ml of a 10 mg/ml Proteinase K solution was added to the tissue and rocked at 54°C for more than two hours. After complete dissolution of the tissue, 700 ml of buffered (pH 8) phenol:chloroform:isoamyl alcohol (24:24:1) were added, the tubes vortexed briefly and centrifuged for 10 minutes at 14k. The supernatant containing the DNA was transferred into a new tube and the DNA precipitated with isopropanol. After spinning, the supernatant was discarded, 70% ethanol was added and centrifuged again. The supernatant was discarded, the DNA air dried and finally resuspended in 100 µl TE-8. 10 ml of DNA prepared above were digested overnight at 37°C in a 30 µl reaction mix containing 10X buffer, 1 mg/ml BSA, 0.001 M spermidine, restriction enzyme and water. mouse enzyme buffer fragments Lmx1b 3’ LacZ BamHI low conc. NEB BamHI buffer 12 kb 8 kb Lmx1b KO -/- BamHI low conc. NEB BamHI buffer 12 kb 4.3 kb

Table 6: Enzymes, buffers and fragments for Southern genotyping

2.4.4.1.3 Gel electrophoresis and Southern transfer 3 µl of loading dye were added to the reaction mix from above and run slowly on a 1% agarose TAE gel containing 3 µl/100 ml ethidium bromide. When completed, a photograph was taken of the gel marked by a fluorescent ruler under a UV lamp. After 30 minutes of depurination in 0.1 N HCl, the gel was washed in water for 2 minutes, trimmed, washed in 0.4 N sodium hydroxide for 20 minutes and put upside down on a

53 Materials and Methods transfer apparatus containing a sponge soaked in 0.4 N sodium hydroxide, Whatman paper and a charged nylon membrane (HyBond N+). After several hours, the membrane was neutralized by two washes in 2X SSC pH 7.0, wrapped in plastic wrap and UV crosslinked.

2.4.4.1.4 Preparation of the probe RediprimeII – a random prime labeling system from Amersham Pharmacia Biotech Inc. (NJ, USA) was used for radioactive labeling of the probe according to the protocol supplied by the Manufacturer. Briefly, the DNA was diluted to a concentration between 2.5 and 25 ng in 45 µl TE-8 buffer, denatured in boiling water, cooled on ice and added into the reaction tube. 5 µl of 32P dCTP (50 µCi) was added and incubated at 37°C for 10 minutes. The reaction was stopped with 5 µl of 0.2 M EDTA. After denaturation at 95°C, a quick spin column from Qiagen was used to remove free nucleotides. The radioactivity was measured in a scintillation counter and the volume of probe needed for hybridization calculated accordingly.

2.4.4.1.5 Hybridization For prehybridization, the nylon membrane was kept rotating in a tube containing Rapid- hyb buffer from Amersham Pharmacia Biotech Inc. at 65°C for at least 15 minutes. The radioactive probe (60x105 cpm/3 ml) was boiled for 5 minutes and added into the tube. After hybridizing overnight at 65°C, two washes at 65°C with 1X SSC containing 1% SDS and 5X SSC with addition of 1% SDS removed excess probe and reduced the background.

2.4.4.1.6 Imaging and analysis Radioactively labeled DNA was detected on a phosphorimager screen which is sensitive for ß-particles or on a photographic film was exposed overnight at -80°C to reveal the position of the bands. Lmx1b 3’LacZ transgenic mice showed a band at 8 kb due to an additional cut site, whereas the band of wild-type animals was located at 12 kb. The mutant band in Lmx1b KO -/- using 5’ external probes could be found at 12 kb length whereas the wild-type one was at 4.3 kb.

54 Materials and Methods

2.4.4.2 PCR Introduction: DNA can be amplified by the use of polymerase chain reaction, discovered by Karry Mullis in 1986. This standard technique was performed in a Perkin Elmer Cetus DNA Thermal Cycler. Doubled stranded DNA is separated by heating to 95°C and cooling down to 60°C allows oligonucleotides called primers to anneal to the region to be amplified. A temperature resistant enzyme, Taq (Thermus aquaticus) polymerase, extends these oligonucleotides complementary to the DNA sequence of interest at 72°C. Then these newly formed DNA duplexes are separated again by heat. Repeated cycles of cooling for the primers to hybridize, synthesis of new strands by the Taq polymerase and melting amplify DNA quickly in vitro (Lodish et al. 2001).

2.4.4.2.1 PCR for LacZ Lmx1b 3’LacZ mice were genotyped either by Southern blotting or by PCR. The latter method had the disadvantage of only showing that mice were positive or negative for LacZ but did not distinguish between homozygous and heterozygous animals. The time saving PCR technique was used if the mere presence of LacZ needed to be checked or to genotype mice from matings that could not give birth to homozygous offspring according to Mendelian rules.

The assay contained 1 µl DNA from a tail or yolk sac preparation, 15.25 µl H20dd, 2 µl of 2.5 mM dNTPs, 2.5 µl of 10X buffer, 2 µl of 5’ primer, 2 µl of 3’ primer and 0.25 µl Taq polymerase. LacZ1(+): 5’-GCATCGAGCTGGGTAATAAGGGTTGGCAAT-3’ LacZ2(-): 5’-GACACCAGACCAACTGGTAATGGTAGCGAC-3’

Table 7: Primer sequences for LacZ PCR

Start Denaturation Annealing Synthesis End Cycles 5’ at 94°C 45’’ at 95°C 45’’ at 59°C 60’’ at 72°C 7’ at 72°C 35

Table 8: Cycle settings for LacZ PCR

Analysis on a 2.5% TAE gel showed a band of 800 bps for homozygous and heterozygous animals containing a LacZ insert. In wild-type animals, this band was missing.

55 Materials and Methods

2.4.4.2.2 PCR for sex determination In order to determine the gender of animals, PCR was performed under the following conditions: The assay contained 3 µl DNA from a tail or yolk sac preparation, 37 µl H20dd, 5 µl of 2 mM dNTPs, 5 µl of 10X buffer, 1 µl of forward primer (400 ng/µl), 1 µl of reverse primer and 0.5 µl Taq polymerase. Primers sequences were a kind gift from R. R. Behringer’s laboratory. XYFWD(+): 5’-TGAAGCTTTTGGCTT TGAG-3’ XYRVS(-): 5’-CCACTGCCAAATTCTTTGG-3’

Table 9: Primer sequences for X/Y PCR

Start Denaturation Annealing Synthesis End Cycles 5’ at 94°C 30’’ at 94°C 45’’ at 50°C 45’’ at 72°C 5’ at 72°C 40

Table 10: Cycle settings for X/Y PCR

The PCR product was analyzed on a 2.5% TAE gel containing ethidium bromide. Two bands could be distinguished: females (XX) showed a 300 bp fragment, males (XY) two fragments of 300 bps and 280 bps.

56 Materials and Methods

2.5 Whole mount and histological analysis 2.5.1 X-gal staining

Introduction: The LacZ reporter gene cloned into the transgenic mice described in this study encodes for an enzyme termed ß-galactosidase. Its natural function is to split the disaccharide lactose into the monosaccharides glucose and galactose. In science, X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside), a colorless analog of lactose is used to detect regions where the LacZ gene is active. Hydrolysis of X-gal by Figure M2.10: X-gal molecule: 5-bromo- ß-galactosidase yields a product of intense blue color 4-chloro-3-indolyl-ß-D-galactoside (from Sigma catalogue) (Lodish et al. 2001).

2.5.1.1 X-gal staining on whole mounts

2.5.1.1.1 Fixation After dissection of embryos and rinsing in PBS, fixation was performed in 0.2% glutaraldehyde, 2% formalin, 5 mM EGTA (ethylene glycol bis-(2-aminoethyl ether)) pH

8.0, 2 mM MgCl2 in 0.1 M phosphate buffer pH 7.3. The fixative was always made shortly before use as thorough glutaraldehyde fixation was an important step to obtain good staining results. Fixation time was chosen specifically for each embryonic stage and for tissue culture: Stage Time Conditions E7.5 30''-10' at room temperature E8.5 5' – 15' at room temperature E9.5-11.5 15' – 30' at room temperature > E11.5 45'-60' at room temperature, rotating Tissue culture 4' at room temperature Table 11: Embryo and tissue fixation times for X-gal staining

Mammary glands were completely submerged in fixation solution in Quadriperm dishes cooled on ice and fixed for 30 minutes. Additionally, for fixation of older stages, the ice bucket was put on a rotator moving at low frequency.

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2.5.1.1.2 Washing and equilibration Washing in equilibration solution containing 0.1% sodium deoxycholate, 0.2% Nonidet P-

40 from ICN Biomedicals Inc. (Aurora, Ohio, USA), 2 mM MgCl2 in 0.1 M phosphate buffer pH 7.3 removed the fixative and adjusted the pH. The following times and frequencies for washing with equilibration solution were used: Tissues/Stage Time Mammary glands 3 X 10' Tissue culture 2 X 2-5' Younger (E10.5) embryos 3 X 10' Table 12: Equilibration time in different tissues and embryonic stages

2.5.1.1.3 X-gal staining Staining was performed at 37°C on a rotator or at room temperature protected from light. Staining time varied depending on the fixation, on the kind of tissue and on the genetic properties. Most common were 12-18 hours of staining for tissue from heterozygous mice and only half the time for cells carrying two alleles for LacZ. The progress of staining was controlled under the dissecting microscope and proceeded with the protocol when judged complete. As over staining does not give any background, preferably samples were stained longer.

2.5.1.1.4 Washing and storage X-gal staining solution was removed, filtered if contaminated with cellular particles, stored at 4°C and reused. After washing three times in PBS for 10 minutes, the tissue was stored in 4% paraformaldehyde in PBS or in 70% ethanol at 4°C. Alternatively, clearing in BABB followed as described below.

2.5.1.2 Clearing techniques Staining pattern of inner parts of embryos was often covered from sight as the tissue was not clear after staining with X-gal or BCIP/NBT. In order to examine expression in detail and take excellent photos, embryos were cleared with two techniques:

2.5.1.2.1 In benzyl alcohol and benzyl benzoate (BABB) Dehydration was performed in ethanol series (50% - 70% - 85% - 95% - 100% each step lasting 10 minutes to one hour, depending on the size of the tissue; 100% over night at 4°C for embryos or 3 hours at room temperature for mammary glands) as quickly as possible as

58 Materials and Methods after X-gal staining diffusion of the color product could occur in alcohol. On the other hand, the tissue only became entirely transparent if dehydration was complete. Benzyl alcohol (Sigma-Aldrich Corp., St. Louis, MO, USA) and Benzyl benzoate (Sigma- Aldrich Corp., St. Louis, MO, USA) were mixed in a ratio of 1:2. Only glass equipment was used for all steps where BABB was involved. Clearing time ranged from several hours to several days rotating at room temperature.

2.5.1.2.2 In glycerol Samples were dehydrated and cleared in increasing concentrations of glycerol in PBS: 30% - 50% - 70% - 80% for 15 minutes to 1 hour each step, depending on the embryo size. 0.05% Sodium azide was added for samples intended for long-term storage.

2.5.1.3 Staining of frozen sections with X-gal

2.5.1.3.1 Fixation After dissection, the tissue was rinsed in PBS and fixed immediately in X-gal fixative (in

0.2% glutaraldehyde, 2% formalin, 5 mM EGTA pH 8.0, 2 mM MgCl2 in 0.1 M phosphate buffer pH 7.3). Time was adjusted according to the kind of tissue: Stage Time Temperature E7.5 30'' - 10' at room temperature E8.5 5' - 15' at room temperature E9.5-11.5 15' - 30' at room temperature > E11.5 45' - 60' at room temperature, rotating Mammary glands 30' on ice (rotating) Tissue culture 4' at room temperature Table 13: Fixation times for frozen section After fixation, the tissue was washed three times in PBS for 10 minutes.

2.5.1.3.2 Dehydration and embedding Samples were dehydrated in 15% sucrose in PBS at room temperature until they sank to the bottom followed by complete dehydration in 30% sucrose in PBS overnight at 4°C, rotating. Sucrose solution was replaced by prewarmed embedding media (7.5% gelatin, 15% sucrose, 0.05% sodium azide in PBS) and the embryos equilibrated at 37°C. Peel-A-Way embedding molds from Fisher Scientific (Pittsburgh, PA, USA) were filled by half with embedding media and let sit until the media had hardened. Then molds were filled up to the top and the tissue or embryos transferred into the mold. Under the dissecting microscope the position of the embryos was adjusted. Molds were frozen on a metal rack cooled by liquid nitrogen in a Styrofoam box and stored in a freezer at –80°C.

59 Materials and Methods

2.5.1.3.3 Sectioning Frozen sections were cut on a Leica CM3050 cryomicrotome equipped with single use microtome blades from Fisher Scientific (Pittsburgh, PA, USA). The temperature of samples was equilibrated and then frozen blocks were glued onto chucks with Tissue-Tek OCT (VWR, West Chester, PA, USA). Embryos and mammary glands were sectioned at temperatures between –20° and –25°C and adult brain at –15° to –20°C. Thickness of the sections ranged between 5 µm and 50 µm and depended on the kind of tissue and on the purpose. For standard X-gal staining 18 µm or 20 µm were used, for brain sections 30 µm, for mammary glands as little as 5 µm and for antibody staining 20- 25 µm. Sections were put on Superfrost Plus Slides (Fisher Scientific, Pittsburgh, PA, USA), dried at room temperature for one hour and stored at –20°C pending further analysis.

2.5.1.3.4 Postfixation and equilibration After slides had dried or thawed, they were rehydrated in PBS for 10 minutes and postfixed in 2% paraformaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 in PBS for 12 minutes at room temperature, rinsed in PBS briefly and then in equilibration solution twice for 10 minutes each step.

2.5.1.3.5 X-gal staining and washing Slides were stained in X-gal for several hours up to several days at room temperature or at 37°C protected from light. Progress of staining was controlled regularly under the dissecting microscope. Then slides were washed in PBS several times, postfixed optionally in used fixative and counter stained.

2.5.1.4 Other staining techniques and mounting

2.5.1.4.1 Counterstaining with hematoxylin and eosin Hematoxylin is a base and stains acidic nuclear elements in purple to blue color whereas acidophilic eosin stains the cytoplasm. After staining slides with X-gal, counter staining with hematoxylin and eosin or with eosin only was performed. 0.5 g Eosin Y from Sigma-Aldrich Corp. (St. Louis, MO, USA) was dissolved in 500 ml 70% ethanol, 5 ml glacial acetic acid added and stirred overnight. Hematoxylin I was purchased from Richard-Allan Scientific (Kalamazoo, MI, USA).

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2.5.1.4.1.1 Eosin counter stain After dehydration in ethanol series (30% - 50% - 70% each step for 30 seconds to 1 minute), slides were counterstained in eosin for 15 to 30 seconds and completely dehydrated (70% - 85% - 95% for 1 to 2 minutes each step). Two changes in 100% ethanol for 5 minutes were followed by two changes in Histoclear II from National Diagnostic (Atlanta, Georgia, USA) for 10 minutes and cover slipping with Permount mounting media from Fisher Scientific.

2.5.1.4.1.2 Hematoxylin and eosin counter stain For nuclear counter staining, slides were rinsed in PBS, stained in Hematoxylin I for 1 to 2 minutes and rinsed under running tap water. Then the steps described in the eosin protocol in 2.5.1.4.1.1 followed.

2.5.1.4.2 Mounting and storage In general two different kinds of mounting media were used:

2.5.1.4.2.1 Non-aqueous mounting media Non-aqueous mounting media was employed to cover samples stained with X-gal and counterstained with eosin and /or hematoxylin, for alcian blue staining and for cresyl violet stained brain sections. After dehydration and clearing, 2-3 drops of Permount or Entellan (Merck, Darmstadt, Germany) were applied on a slide and protected with a coverslip. When the media was dry, slides were cleaned with xylenes or ethanol.

2.5.1.4.2.2 Aqueous mounting media Aqueous mounting media protected samples from drying out after antibody staining. Two different kinds were used: Aquamount (VWR, West Chester, PA, USA) and Gel/Mount (Biomeda; Foster City, CA, USA) and proceeded as described above for Permount. Fluorescent dyes started to diffuse when using Aquamount after a couple of days which they did not when using Gel/Mount. Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA) was employed for the QCPN – Cy3 stained quail-chick chimeric limbs. To ensure samples did not dry out when using Aquamount, coverslips were circled with nail polish.

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2.5.2 Immunohistochemistry

Introduction: Antibody staining is employed to detect and distinguish different molecules in whole mounts or sections. For visualization a secondary antibody conjugated with an enzyme or a fluorescent dye is applied. Confocal scanning of tissue stained with fluorochromes allows analysis of antigen distribution in high resolution and in three dimensions.

2.5.2.1 Standard antibody staining Slides were circled with a PAP pen (Ted Pella Inc., Redding, CA, USA) and the gelatin/sucrose embedding media dissolved in PBS at 37°C for 10 minutes. Then slides were fixed in ice-cold methanol for 20 minutes, dried briefly and blocked in 3SB (0.3% Boehringer blocker from Boehringer Mannheim (Indianapolis, IN, USA) 5% fetal calf serum, 5% goat serum, 1% chick serum, 0.2% Triton X-100 in PBS) for one hour to reduce unspecific binding of the antibodies. Primary antibodies were diluted in 3SB and 100 µl applied on the slides housed in Quadriperm dishes (Heraeus Instruments, Inc; Kendro Lab Products, Newtown, CT, USA) and incubated in a humidity chamber at room temperature for 60 minutes or at 4°C overnight. Slides were washed twice in PBT for 10 minutes followed by 20 minutes of blocking in 3SB. Secondary antibodies were diluted in 3SB, applied and slides incubated in a humidity chamber for 30 minutes at room temperature. For nuclear counter staining, TOPRO-3 (Molecular Probes, Inc., Eugene, OR, USA) was added in a ratio of 1:50 into the secondary antibody solution. After incubation, slides were washed again in PBT for 10 minutes each. In the cases when counterstaining with DAPI (Roche Diagnostics Corporation, Indianapolis, IN, USA) was used, the nuclear marker was added into the PBT washing solution or into the methanol fixative (1:2000 from a 1 mg/ml stock). Slides were mounted as described in 2.5.1.4.2.

2.5.2.2 Variations of standard antibody staining

2.5.2.2.1 Double labeling using antibodies from the same species Introduction: Double labeling with two antibodies made in identical species carry the risk of cross-reactions of the different elements used in the procedure giving false positive or false negative results and therefore need to be controlled precisely.

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Figure M2.11: Illustration of double labeling with antibodies from the same species A. Incubation with the first primary antibody (e.g. rabbit anti-ß-gal); B. Incubation with the first secondary antibody (e.g. goat anti-rabbit Cy3); C. Incubation with normal serum as a source of non-immune IgG from the same host species as the primary antibodies (rabbit). Open binding sites on the first secondary antibody are saturated to prevent binding to the second primary antibody; D. Incubation with an excess amount of unconjugated F(ab) – fragments against the host species of the primary antibody (goat anti-rabbit); E. Incubation with the second primary antibody (e.g. rabbit anti-Lbx1); F. Incubation with the second secondary antibody (e.g. goat anti-rabbit Alexa 488). (Adapted from the Jackson Immuno Research Laboratories catalogue)

Basically, two independent standard antibody staining procedures were performed sequentially with important modifications. After the first staining with application of primary and secondary antibodies had been completed, the open binding sites of the secondary antibody needed to be saturated. Normal serum from the same host species as the primary antibodies was applied to prevent the second primary antibody to bind to the first secondary antibody.

Moreover, as there were still some open binding sites on the first primary antibody, Fab- fragments against the host species of the primary antibody and derived from the same host species as the secondary antibody were used for saturation. After preparing samples this way, the second antibody staining followed (Eichmuller et al. 1996).

2.5.2.2.1.1 First antibody staining procedure Samples were fixed, blocked and stained as described in the standard antibody staining protocol in section 2.5.2.1.

2.5.2.2.1.2 Saturation of open binding sites on the first secondary antibody Following the washing steps after application of the first secondary antibody, slides were incubated in saturation solution (4% rabbit serum, 4% fetal bovine serum and 0.2% Triton X-100 in PBS) in a humidity chamber for 30 minutes at room temperature. Three washes in PBT for 5 to 10 minutes each proceeded.

63 Materials and Methods

2.5.2.2.1.3 Saturation of open binding sites on the first primary antibody Slides were incubated with an excess amount of unconjugated F(ab) fragments to saturate the first primary antibody. Affinipure goat anti-rabbit F(ab)-fragments from Jackson Immuno Research labs (West Grove, PA, USA) were applied in a final concentration of 14 µg/ml diluted in 3SB for one hour. After two washes in PBT for 5 minutes each, samples were incubated in 3SB blocking solution for 10 minutes.

2.5.2.2.1.4 Second antibody staining procedure Application of second primary and secondary antibodies was performed as described in the standard antibody staining protocol in section 2.5.2.1 followed by mounting and storage as described in section 2.5.1.4.2.

2.5.2.2.1.5 Controls Controls are essential in double labeling procedures to carefully monitor whether cross- reactions occur and to check the level of background. In three assays, antibody solution was replaced by serum: a) Omission of the first primary antibody – No staining of the first secondary should be seen. b) Omission of the first secondary antibody – No cross-reaction of the second secondary antibody on the first primary antibody should be found. This controls efficiency of saturation step 2.5.2.2.1.3 c) Omission of the second primary antibody: No staining of the second secondary antibody should be seen.

2.5.2.2.2 K14 antibody staining Myoepithelial cells in mammary glands were marked using a Keratin 14 polyclonal antibody made in rabbit. It was raised against a peptide sequence derived from the C- terminus of mouse keratin 14 (Roop et al. 1984; Hu et al. 2001b). For antigen retrieval the standard antibody staining protocol needed to be modified. A rack of slides was put in a 2l glass beaker filled with 600 ml of 10 mM sodium citrate and boiled in a microwave oven for 20 minutes. Evaporated water was replaced by buffer up to the mark every five minutes. After 20 minutes the slides had cooled down and were washed four times in distilled water, 3 minutes each wash and one wash for three minutes in PBS. Then the standard antibody staining protocol followed, as described in section 2.5.2.1. Keratin 14 antibody was diluted 1:100 in 3SB.

64 Materials and Methods

2.5.2.2.3 Cytokeratin 18 antibody staining Applying anti-mouse secondary antibodies on mouse mammary glands to detect the Cytokeratin 18 monoclonal mouse antibody (Bartek et al. 1991) resulted in high levels of background. Therefore a special technique was used to stain luminal epithelial cells. Antigen retrieval was performed as described for K14 in the preceding section. Then the Vector M.O.M. immunodetection kit from Vector Laboratories, Inc. (Burlingame, CA, USA) was employed for fluorescence staining. Slides were washed 2 times for 2 minutes in PBS. Sections were incubated for one hour in working solution of M.O.M. Mouse IgG Blocking Reagent. After washing, incubation for 5 minutes in M.O.M. Diluent followed. Cytokeratin 18 antibody was diluted 1:30 in M.O.M. Diluent and applied on the samples for 50 minutes at room temperature. Slides were washed two times for two minutes and incubated in working solution of M.O.M. biotinylated anti-mouse IgG reagent for 10 minutes. Then sections were washed again and Fluorescein Avidin DCS applied including 1 µl of TOPRO-3 and 1 µl of DAPI nuclear counterstain. After the final washes in PBS, Gel/Mount was used for mounting.

2.5.3 In situ hybridization histochemistry

Introduction: In situ hybridization is a very sensitive method to detect mRNA, revealing its spatial distribution in the original tissue and furthermore, showing levels of expression. This technique was described first by Pardue and Gall in 1970 (Pardue and Gall 1970). Several variations concerning the kind of the label and the nature of the probe have been established. In this study, oligonucleotides labeled with the hapten digoxygenin were employed. Briefly, RNA probes were added under RNase free conditions on whole mounts (Nieto et al. 1996) or sections, bound to the complementary mRNA strand to be analyzed and were detected with an anti-digoxygenin antibody conjugated with the enzyme alkaline phosphatase. Finally, a color reaction visualized areas of expression.

2.5.3.1 Whole mount in situ hybridization

2.5.3.1.1 Collection of embryos and dehydration

Mouse and chick embryos were collected and dissected in PBSDEPC. Cavities like the forebrain and the roof of the hindbrain were opened to avoid trapping of reagents. Fixation was performed rocking at 4°C in a 4% solution of the crosslinker paraformaldehyde. After washing in PBT, embryos were dehydrated in a series of methanol in PBT (25%, 50%, 75%, 100%, 100%) and stored at -20°C.

65 Materials and Methods

2.5.3.1.2 Pretreatment and prehybridization All of the following prehybridization and hybridization steps were performed under RNase free conditions using autoclaved and DEPC treated solutions and disposable tubes. To enhance penetration and equal distribution of solutions, embryos were gently agitated on a speed rocking platform. After rehydration in a series of methanol in PBT, embryos were washed in PBT and bleached in 6% hydrogen peroxide in PBT for one hour to inhibit endogenous peroxidase activity and washed again. Proteinase K treatment (10 µg/ml) for 10 to 20 minutes, depending on the size of the embryos, digested cellular proteins partially hereby rendering mRNA accessible for the probe. Application of 2 mg/ml glycine in PBT for 10 minutes stopped protein degrading enzyme activity and was followed by morphology preserving postfixation in 4% paraformaldehyde and 0.2% glutaraldehyde for 20 minutes. After washing in PBT, embryos were prepared for hybridization in prehybridization buffer for 1 hour at 70°C.

2.5.3.1.3 Hybridization and posthybridization The solution was removed, fresh prehybridization mix containing 1 µg/ml digoxygenin probe added and hybridized over night at 70°C. Excess probe was washed out in solution 1 (50% formamide, 5X SSC pH 4.5, 1% SDS in

H2ODEPC) (3 times, 30 minutes each at 70°C) and in solution 2 (50% formamide, 2X SSC pH=4.5 in H2ODEPC)(3 times, 30 minutes each at 65°C). RNase treatment was omitted.

2.5.3.1.4 Antibody staining and color reaction TBST washing permeabilized the tissue and inhibited endogenous alkaline phosphatase activity. After preblocking with 10% sheep serum in TBST for 2.5 hours at room temperature, the digoxygenin antibody mix was applied over night rocking at 4°C. To decrease background, embryos were washed for one day in TBST, then 3 times in NTMT alkaline phosphatase buffer (100 mM sodium chloride, 50 mM magnesium chloride, 100 mM Tris pH 9.5, 0.1% Tween-20, 2 mM Levimasole). The color reaction mix was added and the progress of the staining monitored. When judged complete, embryos were washed in NTMT and PBS pH 5.5 and stored in 4% paraformaldehyde.

66 Materials and Methods

2.5.3.2 Section in situ hybridization combined with antibody staining

2.5.3.2.1 Paraffin embedding Embryos were dehydrated in a series of ethanol and xylenes and embedded in paraffin at 60°C. Blocks were sectioned serially on a microtome at 8 µm and put on slides pretreated with glycine/gelatin solution. Storage in a warming chamber at 40°C ensured adherence of the tissue to the slides.

2.5.3.2.2 Preparation and prehybridization The tissue was dewaxed and rehydrated in a series of xylenes and ethanol and postfixed in 4% paraformaldehyde in PBS for 30 minutes. Treatment with Proteinase K for permeabilization was omitted without significant loss of staining (Streit and Stern 2001). Performing this step prevented QCPN anti-quail antibodies from detecting quail epitopes. Slides were washed in SSC 2X pH 7.0 and then in Tris/glycine buffer (0.5 M Tris base, 0.5

M glycine in H2O).

2.5.3.2.3 Hybridization and posthybridization The probe was heated up to 95°C, cooled on ice and applied on the slides in prehybridization buffer containing sodium ions and formamide to stabilize the hybrids. Carrier RNA/DNA, polyvinylpyrrolidone, serum and Ficoll increase the efficiency and specificity of the hybridization and reduce unspecific binding (Hougaard et al. 1997). Coverslipping with parafilm ensured an even distribution of the probe and reduced evaporation during hybridization in a humidity chamber containing SSC 5X and 40% formamide over night at 65°C. Parafilm was removed in 5X SSC, slides were washed in 0.5X SSC, 20% formamide at 60°C and in 2X SSC at room temperature, treated with RNase A (Boehringer, 12.5 µg/ml) at 37°C to destroy single strand RNA and washed again in 2X SSC and in 0.5X SSC, 20% formamide.

2.5.3.2.4 Antibody staining and color reaction Alkaline phosphatase conjugated anti-digoxygenin antibodies (Boehringer Mannheim, Germany) were applied in a 1:4000 dilution in blocking solution (Boehringer Mannheim, Germany) together with undiluted QCPN anti-quail antibodies (DSHB, Iowa City, Iowa) and incubated over night in a humidity chamber containing 2X SSC. Washing 8 times in 1X TBS removed unbound antibodies. Tissue was prepared for color reaction in NTMT alkaline phosphatase buffer (100 mM sodium chloride, 50 mM magnesium chloride, 100 mM Tris pH 9.5, 0.1% Triton X-100) and incubated with

67 Materials and Methods

BCIP/NBT color reaction mix in a box protected from light until staining was judged complete. If the reaction was slow, the color reaction mix was exchanged every 24 hours. Staining was stopped in 1 mM EDTA in PBS. The standard antibody staining followed, applying again QCPN undiluted as primary antibody and using a fluorescent Cy3 goat anti- mouse antibody from Dianova in a 1:200 dilution for detection. Photographs were taken under a standard light source and under a fluorescence microscope and digitally overlaid on a computer.

68 Materials and Methods

2.6 Photography and imaging

2.6.1. Photographs of slides Photographs were taken on an Axioplan 2 microscope from Zeiss equipped with an AttoArc2, HBO 100W lamp for fluorescence connected to an AxioCam linked with fiber optics to a G4 Macintosh computer. Different lenses were used ranging from 5x to 40x and an oil immersion one with 63x magnification. Optionally, digital interference contrast (DIC) was turned on. Another Axioplan microscope was connected to a Hamamatsu camera system.

2.6.2. Photographs of whole mounts Photographs of whole mount embryos and mammary glands were taken under a Zeiss dissecting microscope equipped with either a Hamamatsu color chilled 3CCD camera connected to a Hamamatsu camera controller or with a Zeiss AxioCam connected to a G4 Macintosh computer. For illumination the integrated light source from the Zeiss dissecting microscope table and external cold light were used.

2.6.3. Confocal microscopy Some photographs of samples with fluorescent staining were taken with an Olympus FV500 Laser Scanning Confocal Microscope equipped with three different lasers: Argon 488; Helium-Neon 543 and Helium-Neon 633. The microscope was controlled by and photographs imported with Fluoview software on a PC.

2.6.4. Software All photographs were imported from the digital cameras and worked on using Adobe Photoshop versions 5.0 and 6.0 and Adobe Image Ready version 3.0. Corel Draw 10 was utilized for the final layout of the figures.

69 Materials and Methods

2.7 Solutions, buffers and antibodies 2.7.1. Solutions and buffers

PBS 10x stock solution 80 g sodium chloride (NaCl) 2 g potassium chloride (KCl) 11.5 g sodium phosphate (Na2HPO4) 2 g potassium phosphate (KH2PO4) dissolved in 1 l of distilled water pH 7.4

PBS DEPC

0.1% diethyl pyrocarbonate (C6H10O5) in PBS

50X TAE 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA pH 8.0 to a final volume of 1 l

20X SSC 175.3 g sodium chloride, 88.2 g sodium citrate dissolved in 1 l of distilled water, pH adjusted to 4.5 with citric acid

TE-8 10 mM Tris-HCl pH 8, 1 mM EDTA pH 8

LB broth contains 10 g/l Bacto-tryptone, 5 g/l Bacto-yeast extract, 10 g/l sodium chloride TB broth contained 12 g/l Bacto-tryptone, 25 g/l Bacto-yeast extract, 4 g/l in phosphate buffer

(0.017 M KH2PO4, 0.072 M K2HPO4).

X-gal stock solution 25 mg/ml in 70% dimethylformamide, 30% water stored at –70°C

X-gal staining solution 1 mg/ml X-gal 5 mM K ferricyanide (FW: 329.2) 5 mM K ferrocyanide (FW: 422.4) in equilibration solution

2.7.1 Solutions and buffers

70 Materials and Methods

For In Situ Hybridization:

10X TBS

8 g sodium chloride, 0.2 g potassium chloride, 25 ml Tris HCl 1 M pH 7.5, 75 ml H2O

TBST 1X TBS, 2 mM Levimasole, 0.1% Tween-20

Reaction mix

Stock solution: BCIP (5-bromo-4-chloro-3-indolyl phosphate) 50 mg/ml in H2O, NBT (4- nitro blue tetrazolium chloride) 75 mg/ml in 70% dimethylformamide Working solution: 3.5 µg/ml BCIP, 4.5 µg/ml NBT

Prehybridization buffer for section ISH 40% formamide, 5X SSC, 1X Denhardt’s, 1% t-RNA (Sigma, 10 µg/µl stock), 1% hering sperm DNA (Sigma, 100 µg/µl stock), 1% probe

Denhardt’s 100X 20 g Ficoll (Type 400, Pharmacia) 20 g Polyvenylpyrolidone 20 g bovine serum albumin (Fraction V, Sigma) in 1l H2O DEPC treated and filtered

Boehringer blocking solution-stock

10% Boehringer blocking agent in H2O, dissolved 60°C

Blocking solution (for QCPN antibody staining) PHT = 1% bovine serum albumin in PBT

Prehybridization solution for whole mount ISH All components were made up with H20DEPC and filtered 50% formamide, 5x SSC pH 4.5 (use citric acid to pH), 50 µg/ml yeast RNA, 1% SDS, 50 µg/ml heparin, 1µg probe/ml

Digoxygenin antibody mix 20 mg embryo powder were incubated in 20 ml TBST for 30 minutes at 70°C and then cooled on ice. 200 µl fetal calf serum and 10 µl alkaline phosphatase conjugated anti- digoxygenin antibody (Boehringer Mannheim, Germany) were added, the mixture incubated for more than one hour on ice and centrifuged before use for 10 minutes at 4500 rpm at 4°C.

Embryo powder Stage 32 chick embryos were sacrificed, the heart removed and homogenized in a small amount of ice cold PBS. Four times the volume of ice cold acetone were added, the mixture vortexed and put on ice for 30 minutes. After spinning at 10.000 g for 10 minutes at 4°C, the supernatant was discarded and the pellet rinsed in ice cold acetone, air dried on a filter paper and grinded to powder in a mortar with a pestle.

71 Materials and Methods

2.7.2 Antibodies and counterstains

2.7.1.1 Primary antibodies MK 14 (Covance Inc., Princeton, New Jersey, USA) 1:100 dilution Cytokeratin 18 = K18 (PROGEN Biotechnik GmbH, Heidelberg) 1:30 dilution WT (C-19): sc-192 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA) WT (F-6): sc-7385 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA) MyoD (M-318): sc-760 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA) myogenin (M-225): sc-576 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA) QCPN (Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA) monoclonal mouse IgG1 antibody; 1:10 ß-gal (Cortex Biochem, Inc., San Leandro, California, USA) 1:500 polyclonal IgG Lbx1 (kind gift from Martyn Goulding) rabbit antibody 1:100

2.7.1.2 Secondary antibodies Alexa 488 goat anti-rabbit (Molecular Probes, Inc., Eugene, OR, USA) 1:700 Alexa 568 goat anti-rabbit (Molecular Probes, Inc., Eugene, OR, USA) 1:500 Cy3 conjugated goat anti-rabbit secondary antibody (Chemicon, Temecula, CA, USA) 1:500 Cy3 conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany) 1:400 Alkaline phosphatase conjugated anti-digoxygenin antibody (Boehringer Mannheim, Germany) 1:4000

Vector M.O.M. immunodetection kit (Vector Laboratories, Inc., Burlingame, CA, USA)

2.7.1.3 Nuclear counter stains TOPRO-3 iodide (Molecular Probes, Inc., Eugene, OR, USA) (642/661) DAPI (Roche Diagnostics Corporation (former Boehringer Mannheim), Indianapolis, IN, USA)

2.7.2 Antibodies and counterstains

72 Results

3. Results 3.1 Lmx1b 3’LacZ mice 3.1.1 Genotyping

Genotyping of Lmx1b 3’LacZ mice was performed either by PCR or by Southern blotting Lmx1b KO mice were genotyped by Southern blotting.

3.1.1.1 Genotyping by Southern blotting

3.1.1.1.1 Lmx1b 3’LacZ mice Genomic DNA from tail preparation or from yolk sacs was cut with BamHI. Southern blots of mutant Lmx1b 3’LacZ mice (see Fig. 1.1) showed one band at 7.5 Kbps whereas the wild-type band appeared at 11.1 Kbps after hybridization with a 700 bps fragment from the 47BI construct. The mutant fragment was shorter due to an insertion of an additional cut site for BamHI within the construct corresponding to information derived from the DNA sequence.

3.1.1.1.2 Lmx1b 3’LacZ mice crossed with Lmx1b KO mice Lmx1b 3’LacZ -/- mice were crossed with Lmx1b KO +/- mice which resulted in offspring in which one allele contained the LacZ insert and the other one was wild-type (LacZ/WT) or partially deleted (LacZ/KO). Genomic DNA was cut with BamHI and processed for two Southern blots, using the probe 47BI to detect a LacZ insert, displayed in Fig. 1.3 (panel A), and the probe 47B2 to visualize a deletion, shown in Fig. 1.3 (panel B). As described in the Southern blotting section for Lmx1b 3’LacZ mice, LacZ inserts were found at 7.4 Kbps and wild-type bands at 11.1 Kbps (Fig. 1.3 panel A). 47B2 was a 5’ prime external probe labeling a 12 Kbps mutant band (Fig. 1.3 panel B, lanes 1, 2 and 4) and a 4.3 Kbps band for wild-types or LacZ inserts (Fig. 1.3 panel B, lanes 3 and 5) (Chen et al. 1998a). In Fig. 1.3, animals in lanes 1, 2 and 4 carried the genotype Lmx1b LacZ/deletion and animals in lanes 3 and 5 were characterized by Lmx1b LacZ/wild-type. Southern blotting confirmed that all offspring from crossing with homozygous Lmx1b 3’LacZ mice contained one LacZ allele.

73 Results

3.1.1.2 Genotyping by PCR

3.1.1.2.1 Genotyping of Lmx1b 3’LacZ mice PCR for Lmx1b 3’LacZ did not distinguish between homozygous and heterozygous animals as primers were directed against the LacZ insert and validated its presence or absence. Both, LacZ/LacZ and LacZ/WT revealed an amplified product of 800 bps of length. In wild-type animals this band was absent (Fig. 1.2).

3.1.1.2.2 Sex determination of embryos To analyze the gender of embryos during embryonic mammary gland examination, yolk sacs were prepared for PCR. Primers recognized sequences on X and Y chromosomes showing one band in female (XX) animals at 300 bps and an additional band at 280 bps in male (XY) animals (Fig. 1.4).

3.1.2 Phenotype, expression level and localization

3.1.2.1 Phenotype Whole mount and histological examination of Lmx1b 3’LacZ did not reveal an overt phenotypical alteration, neither in heterozygous nor homozygous animals during all embryonic stages, at birth or during further development. The genotype of the progeny was distributed according to Mendelian rules. A colony of homozygous animals was maintained hereby facilitating breeding to other lines.

3.1.2.2 Expression level of ß-galactosidase Expression levels of LacZ were high and proved to be a sensitive tool to examine expression and changes of expression on a cellular level. During fetal development, whole mount embryos containing a LacZ insert and stained with X-gal reacted quickly whereas wild-type embryos from the same litter stained in the same assay did not show any color reaction. Some embryos stained faster than others. To compare the level of expression in heterozygous and homozygous embryos, Lmx1b 3’LacZ +/- and -/- limbs at E10.5 were fixed, embedded, and stained with antibodies directed against ß-galactosidase, the translation product of the LacZ gene. Samples were processed under the same conditions in the same assay to allow comparison. Photographs were taken using the same exposure times. Expression of ß-galactosidase was two times stronger in LacZ/LacZ embryos (Fig. 1.5 A) than in LacZ/WT (Fig. 1.5 B). Furthermore, to obtain the same level of staining,

74 Results sections from homozygous tissue were stained only half the time than sections from heterozygous animals.

3.1.2.3 Localization Comparison of expression patterns obtained from Lmx1b 3’LacZ mice to previously published (Riddle et al. 1995; Vogel et al. 1995; Chen et al. 1998a; Yuan and Schoenwolf 1999; Adams et al. 2000) in situ hybridizations showed no differences and corresponded to what is known about Lmx1b expression. As the ß-galactosidase detection methods were more sensitive, new areas of expression have been found. Lmx1b is a transcription factor and therefore expected to be localized in the nucleus. To determine the position of ß-galactosidase within the cell, a section of a limb was stained with a Cortex ß-galactosidase antibody (Fig. 1.6 C) and nuclei were counterstained with TOPRO-3 (Fig. 1.6 D) detecting double-stranded nucleic acids (Schejter and Wieschaus 1993). High power magnification images scanned in the confocal microscope were analyzed. The dorsal part of E11.5 limbs showed that only nuclei were labeled by the antibody but not the cytoplasm (Fig. 1.6 E). Besides, on sections of cells with basal localization of the nucleus stained with X-gal and acidophilic eosin (cytoplasm), only the nucleus was labeled with X-gal (Fig. 2.14 I; Fig. 2.13 L) while the luminal cytoplasm was devoid of the X-gal reaction product. Taken together, Lmx1b 3’LacZ mice did not display an obviously altered phenotype. Detection of ß-galactosidase, located in the nuclei of cells only, was two times stronger in homozygous animals than in heterozygous mice. Staining of embryos corresponded to known expression patterns.

3.1.3 Intercrosses of Lmx1b 3’LacZ/LacZ X Lmx1b KO +/- mice

Breeding mice homozygous for the reporter gene LacZ to heterozygous knockout mice resulted in offspring with the genotype Lmx1b 3’LacZ/KO. Exons one and two including the first LIM domain were present in one allele. The other allele contained the complete Lmx1b gene followed by the LacZ construct. It was to be determined whether insertion of LacZ reduces significantly expression of Lmx1b as only one modified functional allele was present. Mice were examined for phenotypical alterations. Litters were phenotypically normal at birth and neither abnormal bending of the limbs nor additional foot pads or cranial defects were detected. Examination of live mice after two months did not show any obvious defects. Corneal opacity or other eye phenotypes were not detected.

75 Results

3.2 Detailed analysis of Lmx1b expression in Lmx1b 3’LacZ transgenic mice In this study, expression of the LIM homeodomain transcription factor Lmx1b was examined by X-gal staining in Lmx1b 3’LacZ mice carrying a LacZ reporter gene under the control of the endogenous Lmx1b locus without disrupting endogenous Lmx1b expression. Mice did not exhibit an alteration of phenotype. Detection methods of ß-galactosidase by antibody and X-gal staining were sensitive, allowing detailed analysis even in single cell resolution. No differences were found between staining in this study and what has been described in literature. X-gal did not stain wild-type embryos. Therefore, Lmx1b expression and X-gal staining will be used as synonyms for description of patterns during embryonic development.

3.2.1 Expression of Lmx1b in the murine limb

The following stages were examined on sections, stained with ß-galactosidase antibodies, X-gal and were counterstained with eosin, hematoxylin: E9.5, E10.5, E11.5, E 12.5, E13.5, E14.5, E15.5, E17.5 and newborn. Whole embryos were analyzed from E7.5 to E15.5.

3.2.1.1 Lmx1b expression during embryonic limb development Lmx1b is known as a marker for the dorsal limb bud mesenchyme (Parr et al. 1993; Riddle et al. 1995; Vogel et al. 1995). Expression has not yet been described completely in mesenchyme and in differentiating tissue. In this study, Lmx1b 3’LacZ mouse embryos of all stages were analyzed. Limbs of all embryonic stages revealed staining (Fig. 2.1). By E8.5 and E9.5, expression levels of ß-galactosidase appeared to be low as staining times were prolonged in comparison to later stages. First expression in limbs was detected at E9.5 (Fig. 2.1 C) after induction of limb buds being restricted to the dorsal limb bud mesenchyme (Fig. 2.2 M). A sharp boundary was formed towards the ventral side. In addition to the demarcation of the dorsal limb bud mesenchyme, a second boundary was formed towards proximal (Fig. 2.2 A, B, D; Fig. 2.4 C). Detailed description of the boundary can be found in section 3.2.1.3. Only the ectoderm dorsal of the neural tube was stained from E9.5 onwards, persisting at least until E15.5 (Fig. 2.1; Fig. 2.2 L), but not within the limb (Fig. 2.2 A, D; Fig 2.1 B-H). Expression within the region of the paraxial mesoderm was not observed. During differentiation of the mesenchyme, Lmx1b expression remained restricted to the dorsal part throughout all stages (Fig. 2.2 A-H). Antibody staining for ß-galactosidase (Fig. 2.2 B) and X-gal staining yielded exactly the same pattern (Fig. 2.2 C). Most cells and structures

76 Results within the dorsal part of the limb were positive, but not all. Axons remained unstained (Fig. 2.2 D, Fig. 2.5 P-S) as well as other cells mainly found in proximal dorsal regions of the limb at earlier stages. This will be analyzed in detail in section 3.2.1.5. Counterstaining of X-gal stained limbs with hematoxylin and/or eosin helped to distinguish between the different kinds of tissues. Nuclei marked with X-gal could be clearly separated from nuclei stained with hematoxylin only. All cells in the dorsal part seemed to be stained in whole mount limbs in Fig. 2.1 at embryonic stages E9.5 (C), E10.5 (D), E11.5 (E), E12.5 (F), E13.5 (G) and E14.5 (H). Sections (Fig. 2.2) exhibited a more distinct pattern at E11.5 (D), E13.5 (F, G), E15.5 (J) and newborn (H). In almost all kind of tissue staining was found, except for invading axons and undifferentiated and differentiated muscle cells (section 3.2.1.5). Chondrogenic zones, tendons and cells within muscle blastemas were stained (Fig. 2.2 I). All tissues derived from the somatopleure like chondrocytes, stationary mesenchyme or connective tissue were found to express Lmx1b. A gleno-humeral joint at E15.5 (Fig. 2.2 K) exhibited staining in part of the cartilage and in periarticular tendons. In general, X-gal staining in chondrogenic zones and cartilage was low compared to all other stained structures on the same section. Connective tissue in subcutaneous regions of the dorsal limb was strongly stained (E15.5, Fig. 2.2 J, L). Furthermore, strongly stained connective tissue and tendons separated unstained muscle regions (E15.5, Fig. 2.2 N) and marked dorsal parts in fingers at E17.5 (Fig. 2.2 P). In Lmx1b KO -/-, no finger and toe nails were found (Chen et al. 1998a). Notably, the tissue surrounding the nail root exhibits strong expression (E17.5 in Fig. 2.2 O and in newborns in Fig. 2.3 C).

3.2.1.2 Lmx1b expression in newborn limbs As described above, expression of Lmx1b in the limb of newborn Lmx1b 3’LacZ mice remained restricted to the dorsal part of the limb (distal limb in Fig. 2.3 A and transversal section in D). A horizontal section through the dorsal part of the phalanges (Fig. 2.3 B) exhibited staining in all regions, but not in every single cell. The dermis at the finger nail root and cells under the nail body were stained (distal phalange in Fig. 2.3 C). High magnification of an interphalangeal joint in Fig. 2.3 E demonstrated that articular cartilage expressed Lmx1b up to the dorso-ventral boundary dividing the joint in a dorsal expressing and ventral non-expressing part. Tendons and connective tissue were stained while axons were not (Fig. 2.3). Detailed examination of muscle regions showed expression in the tendons attaching to the muscles (Fig. 2.3 K, G) and in the connective tissue components separating muscle fibers including perimysium and epimysium (Fig. 2.3 G, H, I, L).

77 Results

Staining level within muscle regions was low (Fig. 2.3 G, H, I) compared to other tissue types. Still, a clear restriction to the dorsal part was apparent and the small blue color spots in Fig. 2.3 G, H were not a result of diffusion, as sharp boundaries existed even within weakly stained muscle regions or within one group of muscle fibers. The combination of hematoxylin/eosin and X-gal staining revealed X-gal negative nuclei within muscle groups (Fig. 2.3 I, L). Only a subpopulation of cells within muscle regions was positive for Lmx1b. In Fig. 2.3, photographs (M-O) demonstrated Lmx1b expression in the skin with restriction to the dermal papilla in the base of hair follicles (N, O) and connective tissue in the dermis and hypodermis.

3.2.1.3 Lmx1b expression forms a sharp boundary between dorsal and ventral. Starting at E9.5 with the beginning of limb bud outgrowth, Lmx1b expression was restricted to the dorsal limb bud mesenchyme, maintaining this boundary, an ‘imaginary line’ located exactly in the middle between the dorsal and ventral part of the limb until birth (Fig. 2.4 C). Most likely, this boundary continued to exist throughout life which has not been examined in detail. In addition, another boundary existed in the proximal limb dividing trunk and the proximal part of the limb sharply (Fig. 2.4 C, I; Fig. 2.2 A, D). These boundaries were observed throughout all stages of limb development in whole mount embryos (Fig. 2.1) and on sections (Fig. 2.2 and 2.4). The sensitivity of X-gal staining allowed examination of the boundary at higher magnifications, showing in Fig. 2.4 B (overview in Fig. 2.4 A) that dorsal and ventral were divided precisely from proximal (zeugopod at E13.5 in Fig. 2.4 F) to distal (carpal section in Fig. 2.4 G and within phalanges in H). Even at single cell resolution, the separation is sharp. The division was maintained throughout all stages: E9.5 (Fig. 2.2 M), E10.5 (Fig. 2.2 A-C), E11.5 (Fig. 2.4 A-D), E13.5 (Fig. 2.4 E), E14.5 (Fig. 2.4 L), E15.5 (Fig. 2.2 J) and newborn (Fig. 2.2 H; Fig. 2.3 A).

3.2.1.4 Lmx1b expression does not respect anatomical structures within the boundary In early stages of limb development, cells were still undifferentiated, no visible structures had formed and the boundary between dorsal and ventral went along a straight line (at E10.5 in Fig. 2.2 B, C and at E11.5 in Fig. 2.4 A). When myogenic precursor cells started to differentiate into muscle blastemas, chondrogenic zones formed and tendons took shape, these tissue structures were not present exclusively in the dorsal or in the ventral part of the

78 Results limb, but developed in an overlapping fashion between the dorsal and ventral compartment. Up to now, it has never been determined where expression changes within one tissue element being located both, in the ventral and dorsal part hereby crossing the boundary. In few cases, where tissue elements were located only to a small extent within the boundary, Lmx1b expression respected structures like a forming bone at E13.5 in Fig. 2.4 N. No type of tissue was found to be excluded from expression at the boundary. Expression appeared to follow an imaginary line within the limb located between dorsal and ventral. Half tendons (newborn Fig. 2.3 K), parts of muscle areas (E15.5 Fig. 2.4 M), parts of articular cartilage (newborn Fig. 2.3 E) or part of chondrogenic zones (E13.5 G, H, P) were stained. Lmx1b expression was related entirely to the position of the boundary but not to complete anatomical structures.

3.2.1.5 Myogenic precursor cells lack Lmx1b expression Cells within the limb are derived from several sources. Limb mesenchyme, cartilage, tendons originate from the lateral plate mesoderm, axons invade the limb from the neural tube and myogenic precursor cells are a derivative of the paraxial mesoderm (Chevallier et al. 1977; Christ et al. 1977; Christ and Ordahl 1995). To address the issue whether all cells in the dorsal part of the limb are positive for Lmx1b expression or if there are any exceptions, limbs were analyzed by histology, whole mount in situ hybridization and antibody staining with specific markers.

3.2.1.5.1 Absence of X-gal in dorsal cells Despite the high sensitivity of X-gal staining, it was found that not all cells in the dorsal part of the limb were stained. Counterstaining of X-gal stained sections with hematoxylin and eosin revealed unstained nuclei. Not only complete structures like invading axons in the limb were devoid of staining (Fig. 2.5 P-S), but also single cells located in the region of the premuscle masses (at E10.5 in Fig. 2.5 H; Fig. 2.5 P) and at E11.5 in Fig. 2.4 D. The muscle blastema at E13.5 in Fig. 2.5 I exhibits clearly that only few cells were stained with X-gal but many with hematoxylin and eosin.

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3.2.1.5.2 Whole mount in situ hybridization Whole mount in situ hybridizations for Lmx1b on chick embryos at stages HH24 (Fig. 2.5 K and HH25 (Fig. 2.5 J) exhibited weaker staining in the area of the dorsal premuscle mass. This area is known to express markers for undifferentiated and differentiating muscle cells, hereby lending further support to the idea that cells in the dorsal part of the limb exist, which do not express Lmx1b. As the area showing unstained cells was co- located with premuscle masses, it was interesting to investigate further in myogenic precursor cells (section 3.2.1.5.4).

3.2.1.5.3 MyoD and myogenin antibody staining The description of newborn limbs in section 3.2.1.2 demonstrated that not all cells within the muscle region were positive for Lmx1b whereas connective tissue surrounding the muscles and tendons were strongly stained. It remained unclear whether cells within the muscle region were muscle cells or connective tissue. Antibody staining of muscle areas with two markers of differentiating muscle cells, MyoD and myogenin in Fig. 2.5 N, O was performed. Sections were counterstained with DAPI to visualize all nuclei. Only a subpopulation of cells, approximately 30%, was positive for the differentiation markers.

3.2.1.5.4 Lbx1 and Lmx1b double labeling A dorsal part of the limb stained with a ß-galactosidase antibody and counterstained with TOPRO-3 in Fig. 2.5 L demonstrated the existence of Lmx1b-negative cells in the dorsal limb bud. To determine which kind of cells were negative for Lmx1b, double labeling with antibodies directed against ß-galactosidase (=Lmx1b) and Lbx1, a marker for myogenic precursor cells, was performed on E10.5 (Fig. 2.5 E) and E11.5 (Fig. 2.5 A-D, F, G) mouse limbs. The dorsal and ventral premuscle masses, labeled by Lbx1 (Fig. 2.5 red in F, green in G) were clearly identified and Lmx1b expression marked the dorsal part of the limb (green in F, red in G). Fig. 2.5 A, a higher magnification of the limb at E11.5 in F, showed Lbx1 positive myogenic precursors in the dorsal (top) and ventral (bottom) premuscle mass and Lmx1b staining in the dorsal part. All Lbx1 positive myogenic precursor cells in the dorsal part of the limb were devoid of staining for Lmx1b, standing in line with results from histology and whole mount in situ hybridization.

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3.2.1.6 Axon invasion of the limb and Lmx1b expression Axons invading the limb were devoid of staining as shown in Fig. 2.5 (P-S) at E10.5 and E11.5 and in a section of dorsal part of a newborn limb in Fig. 2.3 (F). Neurons in the lateral motor column (LMC) of the neural tube project axons into the limb to contact muscles. The medial (M) LMC sends axons into the ventral part of the limb and the lateral (L) LMC projects into the dorsal part of the limb (Tsuchida et al. 1994; Tosney et al. 1995). Axons divided at the base of the limb (Fig. 2.3 Q-S) and LMCL entered the

Lmx1b expressing dorsal part and LMCM stayed in the non-expressing ventral part.

3.2.2 Expression of Lmx1b during mammary gland development

Homozygous Lmx1b mutant mice displayed severe defects in embryonic mammary gland development leading to a complete lack of mammary gland at birth (R. L. Johnson, personal communications). Up to now, expression of Lmx1b has never been described in mammary glands and therefore detailed analysis was performed, divided into four phases of development: I. fetal development; II. postnatal development; III. pubertal and postpubertal development; IV. adult development including pregnancy and involution.

Embryonic and adult mammary gland tissue was collected from Lmx1b 3’LacZ mice and stained with X-gal. The following stages were examined in the first three phases: E10.5, E11.5, E12.5, E13.5, E15.5, newborn, P25, P26, P32, P43, P67, P68. For the fourth phase, these timepoints were explored: during pregnancy at day 8, 9, 13, 15; the day of birth and the 10th day of lactation, 4th and 10th day of weaning.

3.2.2.1 Fetal development The first staining was detected at embryonic day 11 when the mammary bud became visible as a downgrowth from the overlying ectoderm (Fig. 2.6 A). Lmx1b expression was detected from the beginning of the formation of the mammary gland primordium. Only part of the future mammary gland cells were positive for the transcription factor. A "ball- shaped" collection of epithelial cells was stained but not the surrounding condensed mesenchymal cells. At E12.5 and E13.5 the lens-shaped structure was stained completely in contrast to the overlying ectoderm (Fig. 2.6 C). Due to rapid proliferation of epithelial cells, the primordium became elongated and the bulb-shaped structure was stained completely at stage E15.5.

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3.2.2.2 Postnatal development At birth the parenchyma was barely developed exhibiting only a few orders of branching. In mice, one primary duct formed by an epithelial layer two cells thick connected secondary ducts lined by a single layer of cells to the outside via the nipple (Richert et al. 2000). Epithelial cells in primary and secondary ducts were strongly positive for Lmx1b (Fig. 2.8 A-C; Fig. 2.9 A-C; Fig. 2.10 A, C). X-gal and H&E counterstaining in Fig. 2.10 D displayed two kind of cells separated by the basal lamina from the stroma: rounded and cuboidal luminal epithelial cells (Fig. 2.10 D) and elongated myoepithelial cells (Fig. 2.10 D). Only nuclei of rounded luminal epithelial cells were stained with X-gal while myoepithelial cells lacked expression. Only a low percentage of the mammary gland fat pad was occupied by the Lmx1b positive parenchyma in postnatal and prepubertal development (Fig. 2.8 A). The stroma consisting of connective tissue, fibroblasts and fat cells (Fig. 2.9 C; Fig. 2.10 A) as well as the proximal lymph node (Fig. 2.9 D) or blood vessels were completely devoid of staining during all stages of development examined (Fig. 2.8).

3.2.2.3 Pubertal and postpubertal development At about three weeks of age, which is the timepoint of the onset of puberty, parenchyma began to respond to elevated hormone levels (Medina 1996). Consequently elongation of the mammary ducts and allometric growth started, driven by mitotic activity in terminal end buds (TEB) where Lmx1b expression was detected (Fig. 2.9 A, B), as well as in the ducts (Fig. 2.10 D, B). No gradients or different levels of expression between already formed and newly forming structures were observed (Fig. 2.9 C; Fig. 2.8 B, C). In general, ducts form when inner cells undergo apoptosis to form a lumen. On sections, cells that were presumably determined to become epithelium and other cells that were programmed for cell death seemed to be stained at the same level. All cells in the inner layer of the terminal end buds were positive for Lmx1b. Between three and six weeks of age, most of the growth occurred and ultimately at about three months of age, the terminal end buds reached the periphery of the fat pad regressing to blunt ended structures (Fig 2.8 C, D). Levels of X-gal staining remained unaltered. The area of Lmx1b expression spread in the same way the parenchyma continued to grow.

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3.2.2.4 Adult development including pregnancy and involution Except for a slight response to the estrous cycle no changes in structure occurred as soon as the mammary gland had fully developed. When mice become pregnant, secretions of estrogen and progesterone and other factors initiate a substantial change in morphology (Medina 1996). Whole mount mammary glands exhibited expansion of staining from day 9 (Fig. 2.8 D), day 13 (Fig. 2.8 E), day 15 (Fig. 2.8 F) to day 18 (Fig. 2.8 G) of pregnancy. Comparison of stage E9.5 and P67 (Fig. 2.8) already showed a striking difference in morphology and staining due to the influence of hormones of pregnancy. An increasing number of epithelial cells positive for Lmx1b started to expand, hereby superseding the fat pad. Massive proliferation was observed during growth from day 9 to day 15 when a high percentage of the area of the fat pad was occupied by the parenchyma. The whole mount mammary glands appeared to be completely stained (Fig. 2.8 F). Details of whole mounts show in Fig. 2.9 D a lymph node devoid of staining, surrounded by spreading ducts and forming alveolar cells (high magnification of (D) in (E) at day 15 of pregnancy). All of the inner cells of the ducts were stained but not the surrounding outer cells in Fig. 2.10 I. Distal of the terminal end buds a layer of cells was negative for Lmx1b (Fig. 2.10 K). Until the day of birth, the adipocyte to epithelial ratio decreased further (Fig 2.8 H) and milk secreting units (lobules) formed, containing alveoli that were partially surrounded by a meshwork of Lmx1b negative myoepithelial cells (Fig. 2.10 J).

At birth, the whole mammary gland was still, like in all other stages before, homogeneously and completely stained (Fig. 2.8 H) but gave a patchy appearance at the 10th day of lactation (Fig. 2.8 I) with stronger staining in thin tissue areas and weaker staining in areas where the tissue was thicker. Repetition of X-gal staining on whole mounts and sections with mammary glands from a different animal and an optimized staining procedure gave exactly the same pattern. Details of whole mounts in Fig. 2.9 show homogenous staining at the day of birth (G) and after weaning (J-L) but only partial staining of secreting lobules at the 10th day of lactation (H, I), depending on the area from where the photograph was taken. All sections were stained, some at high levels (Fig. 2.11 A, B) and some on low levels (Fig. 2.11 C). No rule for the distribution of the staining intensity was found but it seemed that the effect of increased tissue density on fixation played a role as well as the thickness of sections.

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After cessation of pregnancy or weaning, a phase of rapid involution occurred (Fig. 2.8 J, K and 2.11 D-G). Epithelial cells underwent apoptosis and debris was removed by neighboring cells and macrophages. Completion of this process of tissue remodeling nearly brought the mammary glands to a state before pregnancy also with regards to Lmx1b expression (Fig. 2.8 K). X-gal staining of mammary glands decreased in the same way as the epithelia to adipocyte ratio comparing 10th day of lactation (Fig. 2.8 I, 2.9 I, 2.11 A) to the 4th day of weaning (Fig. 2.8 J, 2.9 J, K , 2.11 E) and the 10th day of weaning (2.8 K, 2.9 L, 2.11 F, G). The percentage of cells in ducts expressing Lmx1b during weaning (Fig. 2.11 E, G) appeared lower than during development and pregnancy (Fig. 2.10 B, K).

3.2.2.5 Antibody staining with K14 and K18 Two kinds of cell types in the parenchyma can be distinguished that are separated from the stromal compartment by a basal membrane: 1. luminal epithelial cells surrounding the central lumen and 2. myoepithelial cells that form a sheath around the luminal epithelial cells. Both cell types can be identified using typical markers. Several keratins (e.g. 8, 11, 18, 20, 22) specify luminal epithelial cells (Asch and Asch 1985). In this study keratin 18 (Lane 1982) was used (Fig. 2.7 H). Anti-smooth muscle actin or as employed here, keratin 14 labels contractile myoepithelial cells (Fig. 2.7 G) (Moll et al. 1982; Ivanyi et al. 1992). Mammary glands stained with X-gal and H&E showed that only a subset of the parenchymal cells expressed Lmx1b (Fig. 2.10 D, H), raising the question what kind of cells these were. Antibody staining was performed with keratin 14 (myoepithelial cells in Fig. 2.7 G), keratin 18 (luminal epithelial cells in Fig. 2.7 H) and ß-galactosidase (Fig. 2.7 I, J). Nuclei of confocal images (Fig. 2.7 G, H) were counterstained with TOPRO-3 and fluorescence images in (Fig. 2.7 I, J) with DAPI. Comparison of the luminal marker keratin 18 (Fig. 2.7 H) and ß-galactosidase staining (Fig. 2.7 J) showed an identical staining pattern. The slight difference in the appearance was due to the different imaging techniques. Furthermore, fluorescence images (Fig. 2.7 I, J) and H&E staining (Fig. 2.10 D) indicated that all luminal epithelial cells rather than only a significant fraction were stained.

3.2.2.6 Summary Lmx1b expression was detected first in mammary gland primordia in epithelially arranged cells at embryonic day E11.5, persisting during all stages of fetal, pubertal and adult

84 Results development including pregnancy and involution. Down- or upregulation was not observed. Both, the combination of X-gal and H&E staining and antibody staining showed that Lmx1b expression was restricted to luminal epithelial cells exclusively, whereas myoepithelial cells and cells belonging to the stromal compartment were devoid of expression.

3.2.3 Lmx1b expression in the kidney

During analysis of embryos from Lmx1b 3’LacZ mice, early expression in the developing kidney was detected at E11.5 in part of the mesonephros (Fig. 2.12 D). In contrast to strong expression in chick (see Fig. 4.1 F), staining levels were low. The filtration apparatus (Fig. 2.12 F) at later stages, as shown in a section through the whole kidney at E15.5 (Fig. 2.12 E) revealed expression which persisted throughout all stages of embryonic development and was also visualized in whole mounts (E14.5 in Fig. 2.12 A and at E17.5 in Fig. 2.12 B). After birth, Lmx1b expression was not downregulated but persisted postnatally and in adults (Fig. 2.12 C at P26, adult in G-I). Staining was restricted to the glomerulum in the cortex of the kidney. Up to now it has never been demonstrated which cells of the filtration apparatus express Lmx1b. The Wilms’ tumor gene WT-1 is a podocyte-specific marker (Mundel et al. 1997; Pavenstadt 2000). Therefore antibody staining with WT-1 and ß- galactosidase (=Lmx1b) was performed. Separate labeling with WT-1 (Fig. 2.12 L) and ß-galactosidase (Fig. 2.12 O) antibodies of P25 kidneys exhibited the same pattern and it was hypothesized that both genes are coexpressed. In order to test this, double staining was performed (Fig. 2.12 J, K, M, N) and the confocal image in a high magnification of a glomerulum (Fig. 2.12 N) revealed complete congruence. In all, Lmx1b was shown to be expressed in the kidney as early as E11.5 in the mesonephros, persisting during embryonic development and in adult mice in the filtration apparatus. All WT-1-positive podocytes coexpress Lmx1b in the glomerulum.

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3.2.4 Lmx1b expression in other organs and in adults

Lmx1b expression in mammary glands and in the kidney was described in section 3.2.2 and 3.2.3. It remained undetermined up to now whether other organs exist in which Lmx1b expression has not been described up to now. Therefore, every organ was examined by whole mount X-gal staining and on sections. Moreover, embryos of all stages were stained with X-gal and cleared in BABB to visualize new fields of expression in the mouse.

3.2.4.1 Lmx1b expression at embryonic stages This section deals with analysis of whole mount embryos (see Fig. 2.1) and excludes brain expression. Embryonic day E7.5 was the first stage examined. Several attempts were made with different mice and variation of decisive steps in the staining procedure to rule out a technical problem, but in no case ß-galactosidase activity could be detected. In chick embryos, early detection was reported in the mesoderm of the head process and in the primitive streak at the early neurula stage HH4 (Yuan and Schoenwolf 1999). First expression was found at stage E8.5, not only in the rostral neural folds but also in caudal ones Fig. 2.1 B. Limb expression was found from stage E9.5 on as described in section 3.2.1.1. The ectoderm overlying the neural tube and the paraxial mesoderm was positive for Lmx1b (Fig. 2.1 C) and ectodermal staining persisted in this region. At E10.5, the eye and the pharyngeal arches became stained, and then at E11.5 the mammary gland primordia (see section 3.2.2). From E13.5 on, staining in the developing kidney and of the sprouting whiskers was detected and mammary gland primordia became more prominent.

3.2.4.2 Lmx1b expression in adult and embryonic organs Adult and embryonic organs were collected, stained as whole mount with X-gal in the same assay. The kidney from the same animal was stained as positive control. Additionally, organs were processed for sectioning and stained with X-gal.

3.2.4.2.1 Lmx1b expression in heart, liver, spleen and lung At all ages, no staining was found in the heart (Fig. 2.13 A, D), in the liver (Fig. 2.13 B, E) and in the spleen (Fig. 2.13 C, F). The embryonic lung exhibited staining at E15.5 (Fig. 2.13 G, J). In the adult (Fig. 2.13 H, I, K, L), only few positive cells were detected (K), arranged in clusters of 5 to 10 cells in a row (H, L) and being located in the wall of the primary bronchioles or terminal bronchioles.

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3.2.4.2.2 Lmx1b expression in uterus and stomach Uterus (Fig. 2.14 A, C) and stomach (Fig. 2.14 B, D) from adult animals revealed staining. In both whole mount uteri and on sections of uteri containing embryos (as positive control for the assay), staining was detected but at very low levels compared to the embryo at E13.5. Faint staining was detected in few parts of the smooth muscle (vascularized circular layer, myometrium), but only in a small part of the uterus wall in one section. The placenta was devoid of expression. Staining of uteri containing embryos at E8.5 and E9.5 did not exhibit any staining. In the stomach, only the base of gastric pits was stained in Fig. 2.14 D. To control specificity, organs from wild-type animals were stained and gave exactly the same pattern in the stomach.

During embryonic stages, foreguts exhibited staining at E10.5 (Fig. 2.14 E, high magnification in F) and at E11.5 in Fig. 2.14 G as it has been described in chick the foregut endoderm by (Riddle et al. 1995; Yuan and Schoenwolf 1999). Moreover, no expression was found in the testis at E17.5, and in adults in the bladder and in the small intestine.

3.2.4.3 Analysis of a P26 LacZ/LacZ mouse To explore expression in adult tissue, the fur and the brain were removed from a 26-day- old homozygous Lmx1b 3’LacZ mouse, the peritoneum opened and the whole animal was stained with X-gal. All images refer to Fig. 2.15. Overview (A) of the skull from cranial showed staining in the middle ear (B and high magnification in C), in the meninges (D), the eye (E) and the surrounding tissue and interestingly in the sutures of the skull (F). The overview of the abdomen in (G) revealed no staining in the spleen and liver but the kidneys were strongly positive (G, H) and staining was also detected in the female reproductive system (J): in the ovary and in the uterine tube in (H, J), the ureter (J), in the uterus and in retroperitoneal lymph nodes (K). In skeletal structures, the medial parts of the ribs (L) and spines of the vertebra were stained like the sutures (F). A gland (M) in the lateral neck and the skin (N) were positive for X- gal, in agreement with results from skin sections of the newborn in Fig. 2.3 M-O. Moreover, the brain and the spinal cord exhibited expression.

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3.2.5 Brief summary of Lmx1b expression

In Lmx1b 3’LacZ mice, first expression was detected at E8.5 in the neural folds (Fig. 2.1 B) and when limbs started to bud at E9.5 in the dorsal mesenchyme (Fig. 2.1 C) where it persisted throughout embryonic and, presumably, throughout adult life forming a sharp boundary towards ventral and proximal (Fig. 2.4 A, C, I, K). Lbx1-positive myogenic precursor cells migrating into the limb were devoid of Lmx1b expression. Mammary gland primordia were stained from day E11.5 on (Fig. 2.6 B). Expression was maintained in epithelially arranged cells during fetal development and continued throughout all phases of postnatal life confined to luminal epithelial cells (Fig. 2.7 H, J; Fig. 2.10 D). In kidneys, the mesonephros exhibited staining at E11.5 in Fig. 2.12 D, in the filtration apparatus at E15.5 in Fig. 2.12 E. Lmx1b was shown to be 100% coexpressed in podocytes with WT-1 in a P25 kidney (Fig. 2.12 N). In adult mice, it was demonstrated that heart, lung, spleen and the small intestine were devoid of expression (Fig. 2.13) whereas the embryonic and adult lung (Fig. 2.14) and skeletal elements, the skin and neural tissue displayed staining (Fig. 2.15).

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3.3 Induction of Lmx1b and boundary formation 3.3.1 Lmx1b positive cells maintain a homogenous distribution in a micromass culture

Dorsal limb mesenchyme expresses Lmx1b whereas the ventral part is devoid of expression. To determine whether cells in the two compartments in the limb have different properties on the cell surface or in the extra-cellular matrix (ECM), whole limb mesenchyme from Lmx1b 3’LacZ mice at stage E11.5 was dispersed and analyzed for sorting out phenomena in a micromass culture. Cells were collected and processed as described in section 2.3.2. Incubation times ranged from 2 to 72 hours. All cultures in Fig. 3.1 were derived from the same assay. Controls under the microscope during culture exhibited normal cell morphology and growth speed. Alcian blue staining showed that after 72 hours (Fig. 3.1, panel A), patches of cartilage had formed, giving the same appearance compared to what is known from literature (Ahrens et al. 1977; Maini and Solursh 1991). Additionally, wild-type limb cells were cultured for up to five days to control the culture conditions and stained with Alcian blue resulting in a normal pattern of cartilage formation. The density of cells was increasing steadily in micromass cultures between 2 hours of incubation in (Fig. 3.1 A/F) to 50 hours in (Fig. 3.1 E/J). Photographs were taken from each sample and the distribution compared to cultures from the same origin fixed at different timepoints. Assays of dispersed whole limb mesenchyme cells containing 50% Lmx1b positive and 50% negative cells from Lmx1b 3’LacZ mice did not sort out in micromass cultures after 72 hours of reincubation. Dorsal cells marked by X-gal staining remained in a homogenous distribution with ventral cells. Four different cultures with several samples each presented exactly the same patterning. In a different assay, cultures of mesenchyme containing a high percentage of dorsal cells exhibited identical results. In contrast, when the same limb mesenchyme cells were mixed with one third of 3T3 cells secreting Wnt1 or Wnt7a, sorting out was already observed after 12 hours of culture. Lmx1b/LacZ-positive cells separated further from 3T3 and LacZ negative limb cells after 24 and 36 hours of culture, forming blue stained islands. Staining of 3T3 cells cultured separately did not reveal any X-gal reaction. In all, the control assay exhibited sorting out X-gal positive cells from 3T3 cells. On the contrary, when only limb cells were employed, dorsal Lmx1b-positive cells did not sort out from ventral Lmx1b-negative cells irrespective of the concentration of dorsal cells.

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3.3.2 Ventral proximal mouse limb mesenchyme does not initiate Lmx1b expression when grafted into the Progress Zone.

Ventral proximal mesenchyme is devoid of Lmx1b expression. The next series of experiments were set up to examine whether cells from this regions were still capable of turning on the gene. Therefore blocks of ventral proximal limb mesenchyme collected from Lmx1b 3’LacZ mice at E11.5 were grafted into the Progress Zone of Swiss Webster wild-type mice at E11.5, cultured for 26 hours and stained with X-gal to detect a possible onset of Lmx1b expression. Additionally, a piece of dorsal mesenchyme was grafted onto the contralateral unoperated limb, thereby serving as positive control for staining and cell viability (Fig. 3.2 B). Limbs exhibited growth and no changes in morphology indicating culture conditions were good. In all cases (6/6), grafted ventral limb mesenchyme maintained its non- expressing status (Fig. 3.2 C), while the positive control was strongly stained (Fig. 3.2 B).

3.3.3 No induction of Lmx1b in proximal ventral cells by Wnt7a and FGF8b in the limb

To further test the capability of ventral proximal mesenchyme to initiate Lmx1b expression, the ectoderm was removed from Lmx1b 3’LacZ mouse limbs at stage E11.0. 3T3 cells secreting Wnt7a and/or beads soaked in recombinant FGF-8b were applied to the proximal ventral part of limb mesenchyme and cultured for 26 hours (Fig. 3.3). The embryo parts maintained a good morphology (Fig. 3.3 D). After staining with X-gal, no change of the non-expressing status in the ventral part (14/14) was observed.

Application of Series I Series II Total No expression Percentage ventrally Wnt7a 2 2 4 4 100% FGF-8b 2 1 3 6 100% Wnt7a + FGF-8b 5 2 7 7 100% Total: 14 14 100%

Table 14: Overview Wnt7a and FGF-8b application on cultured mouse limbs

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All limbs in Fig. 3.3 (D, E, G, H) had received a FGF-8b bead and were covered with 3T3- Wnt7a cells. In some cases (5/15), the distal mesenchyme (Fig. 3.3 F) was devoid of staining. 3T3-Wnt7a cells cultured in the same assay and stained with X-gal did not exhibit any color reaction in a control in (I).

3.3.4 Lmx1 - induction of ventral proximal cells in chick

3.3.4.1 Whole mount in situ hybridization Ventral proximal mesenchyme blocks from quail donors at stage HH22-24 were grafted into different locations in the dorsal part of chick host wings at HH20-23 (see grafting scheme 2.2.3.2.5.1 – OP A), reincubated for 8-22 hours and analyzed by whole mount in situ hybridization (this section) or by section in situ hybridization (section 3.3.4.2). All limbs looked normal concerning size, outgrowth and morphology compared with the contralateral limb serving as control. All control limbs were stained homogeneously. In all samples (16/16), examination after whole mount in situ hybridization revealed that ventral grafts located within dorsal Lmx1 expressing mesenchyme maintained their non- expressing status. Grafts were not stained and appeared as a white spot on the dorsal limb (Fig. 3.4). To further analyze a change of expression, limbs were sectioned (Fig. 3.4 C) which exhibited the same result that no change had occurred. Dorsal cells surrounding the graft were not influenced and did not show any up- or downregulation of Lmx1.

3.3.4.2 Section in situ hybridization To explore a change of expression on a cellular level, two samples were analyzed by section in situ hybridization combined with QCPN anti-quail antibody staining to detect donor cells within the dorsal part of the host wing. Ventral proximal Lmx1b-negative quail wing mesenchyme (HH24) was grafted into the middle part concerning proximo-distal and anterior-posterior axes of a chick donor at stage 22 and reincubated for 29 hours. Two images were taken, one with normal light of blue BCIP/NBT Lmx1b staining and the other one with fluorescence (QCPN-Cy3 – red) and overlaid digitally. The graft located within the dorsal limb, was completely surrounded by Lmx1b-positive dorsal mesenchyme (Fig. 3.5 G). Cells migrated within the dorsal part of the limb mainly towards distal but also to proximal positions (Fig. 3.5 H). All grafted quail cells within the graft and all migrating cells kept their non-expressing status (Fig. 3.5 G, H). Besides, all migrating cells remained within the dorsal part.

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3.4 Migration behavior of grafted quail cells in the chick wing 3.4.1 Lbx1 expression in quail and chick

Lbx1, known as a marker for myogenic precursors cells, was employed to visualize areas of myogenic precursor cells in the quail and chick wing. Although many studies exist on Lbx1 (Jagla et al. 1995; Mennerich et al. 1998; Schafer and Braun 1999; Brohmann et al. 2000; Gross et al. 2000), the intensity of staining between the dorsal and ventral premuscle mass has never been compared. Antibody staining and whole mount in situ hybridization served as tools. Analysis of Lbx1 antibody staining (green), counterstained with DAPI (blue) in a quail wing at stage HH23 sectioned in the dorso-ventral plain revealed that the expression area in the ventral premuscle mass is larger than in the dorsal premuscle mass (Fig. 4.1 A). Staining in the middle of the image was background or autofluorescence of erythrocytes similar compared to the control in which the primary antibody was omitted in Fig. 4.1 B. In Fig. 4.1 C, a section through a mouse limb at E11.5 exhibiting migration of myogenic precursors in a dorsal and ventral pathway showed that in mice no difference in intensity of the premuscle masses existed. Whole mount in situ hybridization employing Lbx1 (Fig. 4.1 D, D’, F-I) and Pax3 (E) antisense probes on chick embryos at HH stages 19 (G) to HH25 (E) supported the finding of a stronger ventral premuscle mass marked by Lbx1 expression. Comparison of Lbx1 (Fig. 4.1 D’ and D) and Pax3 (Fig. 4.1 E) expression exhibited a difference between the two markers for myogenic precursor cells in a way that only the ventral Lbx1 domain was stronger but not the one labeled by Pax3. Furthermore, serving as basis for further investigation in the ventral wing, the ventral premuscle mass stained with Lbx1 exhibited a “nose-like” offshot in the ventral proximal posterior part directed towards dorsal proximal.

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3.4.2 Interspecific grafting experiments

In the present study, different grafting experiments from quail to chick were carried out in order to examine the migration behavior of myogenic precursor cells in the outgrowing wing of chick embryos. Mesenchyme blocks from different locations of a quail donor wing were grafted into different positions of a chick host, reincubated for 48 hours and analyzed by QCPN anti-quail antibody staining on sections as shown in figures 3.4.2 I-III. Details about the grafting procedure were described in section 2.2 and more information on operations can be found in section 2.2.3.2.5.

3.4.2.1 Overview of transplantations from x to Y in proximal distal operation in quail chick n= migration migration found in long range migration migration close yes no muscle migration (*d=dorsal) (*v=ventral) OP A ventral dorsal 11 5 4 2 5 5 5 OP B dorsal ventral 6 2 2 2 2 2 OP C ventral homotopic 7 4 3 2 4 2 OP D dorsal homotopic 10 4 5 1 2 2 3 OP E ventral pz 9 7 2 7 (*d)6 (*v)7 OP F ventral central 6 2 1 3 Total: 49 24 15 10 16 Table 15: Overview of interspecific grafting experiments Cells were considered to have migrated if located more than 200 µm away from the grafted block and were not in direct contact with the stationary graft tissue. Single cells less than 200 µm away from the graft could be dispersed during the grafting procedure and were not counted as migrating cells as well as if the mesenchyme block stayed compact and no cells left the graft.

In table 15, analyzed samples were sorted according to the kind of operation and put into one of three categories to judge migration: · ‘migration yes’ if migrating cells were found more than 200 µm away from the graft. · ‘migration no’ if no migrating cells were found or only dispersed cells close to the graft. · ‘found in close muscle’ if cells did not migrate far but were integrated in a forming muscle located close to the graft.

Addition of the numbers in the columns ‘migration yes’, ‘migration no’ and ‘found in close muscle’ add up to the total number (n) in each line. If long-range migration within the limb was detected, this was reflected not only in the column ‘migration yes’ but also in ‘long

93 Results range migration’. The last two columns show in which way migrating cells were directed if migration occurred: ‘distal’ or ‘proximal’. In case of operation E in which grafts were inserted distally, the last columns show the distribution in the dorsal and ventral side instead.

In this table, all grafting experiments were added up, also if no migration was expected due to incompatibilities of the donor-host age relationship or the position of the graft. Detailed analysis of the numbers will be given in the following sections. Thus, a comparison of the percentage of migrating and non-migrating cells cannot be derived from this table but will be given in each section.

The specifications of directions in this study were defined as follows to describe migration within the limb. Proximal means towards the base of the limb, distal towards the tip, ventral towards the Lmx1 negative part and dorsal towards the Lmx1 positive part. Central refers to a field located in the middle of all three axes in the limb. The terms medial, lateral, cranial and caudal were employed if migration in extra-limb regions was described.

3.4.2.2 Dorsal cells stay dorsal, ventral cells stay ventral Myogenic precursor cells migrate into the wing after delamination from the lateral lip of the dermomyotomes (Williams and Ordahl 1994). Premuscle masses in the chick wing become established by HH stage 21 (Schramm and Solursh 1990). A question up to now unresolved is, how myogenic precursor cells move within the outgrowing wing and if there is exchange between the dorsal part marked by Lmx1b expression and the ventral side. To address this question, blocks of mesenchyme containing myogenic precursor cells were grafted into different locations of the wing where the position of cells was determined after 48 hours of reincubation. In all (24 of 24) samples where migration had occurred, cells stayed either dorsal or ventral but did not cross the boundary dividing the Lmx1b expressing and non-expressing area.

3.4.2.2.1 Operation A: ventral to dorsal Two series of experiments were performed where grafts were collected from different proximal positions. If taken from and inserted into the base of the limb close to the trunk, long range migration was found in 0 of 6 samples (Fig. 4.3 H). Grafts portrayed as a compact round structure. Only in two cases, grafted cells became located in a close

94 Results premuscle mass. In this series, hosts and donors were both of the same age, operated at stage HH 21. Cells from mesenchyme blocks dissected from and inserted into more distal positions within the proximal wing exhibited migration in 5 out of 6 samples (Fig. 4.2 A, B and Fig. 4.4 A, B). Grafts were collected from quail donors at HH24 and inserted into chick hosts of HH22, ensuring that the host was 2 HH stages younger than the donor. Most cells migrated distally (Fig. 4.2 B and Fig. 4.4 A), fewer cells took a proximal pathway (Fig. 4.4 B).

3.4.2.2.2 Operation B: dorsal to ventral Cells migrated only in 2 out of 6 cases. In two other samples, quail cells were found only in close muscle blastemas, even though donors were two HH stages older (HH24) in comparison to the hosts (HH22). Grafts were taken from very proximal positions. Most migrating cells were found proximal of the grafts (Fig. 4.2 C), in medial or in caudal (Fig. 4.2 D) positions. In one case, single cells migrated within the ventral premuscle mass (Fig. 4.2 D). In this series, as well as in the homotopic grafting procedure, only little migration was found from grafts collected from very proximal positions.

3.4.2.2.3 Operation C: ventral homotopic Detailed examination of Lbx1 expression pattern exhibited the formation of a ‘nose-like’ structure in the ventral proximal posterior region by myogenic precursor cells from the ventral premuscle mass towards dorsal. To explore if there is exchange of myogenic material between ventral and dorsal in this zone or if some cells migrate along this ‘nose- like’ structure and end up in the dorsal part of the wing, ventral proximal posterior quail mesenchyme was grafted into a homotopic region in a chick host. No cells were detected to cross the boundary and become located in the dorsal part of the wing, while migration along the proximo-distal axis was found in 4 out of 7 samples. As shown in Fig. 4.4 C, grafts were well inserted into the proximal ventral region. Cells displayed strong migration in the distal direction within the ventral premuscle mass (Fig. 4.4 D) and in the proximal direction (Fig. 4.4 C) into the trunk region. Cells in the proximal pathway migrated in a stream of cells around the base of the limb and reassembled in a certain area of the trunk in a position caudal to the graft. The same observations were made in operation B (dorsal to ventral graft in Fig. 4.2 C, D). Only few cells migrated towards medial positions and no cells became located cranially. Fig. 4.2 E shows a graft inserted very proximal within the trunk region staying together as a compact quail cell mass. In one case, the block of quail cells contained not only

95 Results mesoderm but also ectoderm which became incorporated in the ventral host’s ectoderm as described previously by (Schramm and Solursh 1990). In two samples it seemed as if myogenic cells were attracted by the closest forming premuscle mass.

3.4.2.2.4 Operation D: dorsal homotopic In two series, grafts were transplanted from proximal positions close to the base of the wing (series 1) and from proximal positions further within the wing (series 2) of quail hosts into the dorsal proximal wing bud exhibiting migration of cells in only two of seven samples in series 1 and in two of three cases in series 2. Additionally, cells in the third sample of series 2 supplied cells for a muscle blastema close to the graft. Taken together, migration was more probable when grafts were collected from proximal positions located not exactly at the very base of the wing. Distal (Fig. 4.2 H) and proximal (Fig. 4.2 G) pathways were taken by migrating cells staying restricted to the dorsal part of the limb. Distal migration was stronger. Only in one case (Fig. 4.3 F) cells in a HH27 wing migrated into caudal proximal positions located in the trunk region but not within the wing, presumably supplying a premuscle mass of the musculotendinous cuff with myogenic material. Cells migrated into a similar position after a grafting ventral mesenchyme into a central proximal region of a limb (operation F – Fig. 4.3 E). Axons invading the limb from proximal divided a dorsally located graft into two parts (Fig. 4.2 F).

3.4.2.3 Cells grafted distally follow either a dorsal or ventral pathway

3.4.2.3.1 Operation E: ventral into the Progress Zone In this series of experiments, proximal ventral cells were collected from quail wings two HH stages older than their hosts and inserted into the Progress Zone. In all cases, cells were found in at least some distance to the graft, in 7 of 9 samples long-range migration was observed in the dorsal and ventral premuscle mass (Fig. 4.3 A, C). Many cells migrated from the distal tip to proximal regions of the wing, taking either a dorsal or a ventral pathway (Fig. 4.4 F, G) but were never found migrating in a central region. Therefore, cells must have remained on the side they started migrating in and did not exchange pathways between dorsal and ventral.

96 Results

Grafts became located either dorsally (Fig.4.3 A, C), ventrally (Fig. 4.3 B) or in the distal tip of the limb (Fig. 4.4 G). If grafts were not located distally, migration towards proximal and distal could be observed (Fig. 4.3 A). 6 out of 7 samples displayed migration in the dorsal region and cells from all samples (7/7) migrated in the ventral region. Two samples (Fig. 4.4 F, G) were reincubated for 18 hours, all other embryos for 48 hours. The migration behavior was the same, only less intense and for a shorter distance.

3.4.2.4 Formation of segment overlapping muscles After operation E, in one sample cells migrated in dorsal and ventral premuscle masses and supplied a dorso-ventral-segment overlapping muscle blastema with myogenic material from both sides (Fig. 4.3 D). This was the only case examined where quail cells became located in a region at the dorso-ventral boundary.

3.4.2.5 No migration within the chondrogenic zone

3.4.2.5.1 Operation F: ventral to central Ventral mesenchyme blocks containing myogenic precursor cells were grafted into a central zone meaning in the middle of the dorso-ventral, anterior-posterior and proximo- distal axes. Grafted cells integrated in a chondrogenic zone did not migrate (Fig. 4.3 G) unless the graft had partial access to mesenchyme allowing cellular movement. If the graft was not in an optimal central position, migrating cells were found proximally or dorsally. In one case, in which the graft was not collected from ventral but from a central location and inserted into a central position, cells stayed together as a compact mass (Fig. 4.4 H, I).

3.4.2.5.2 Grafts positioned in the chondrogenic zone in other operations In general, when grafts became located within a chondrogenic zone, samples exhibited migration only if the quail cells were not surrounded completely by chondrogenic cells. In several other cases where transplanted cells stayed together in a compact structure and did not migrate, these grafts were completely or partially inserted into a chondrogenic zone.

3.4.2.6 Differences in migration between dorsal and ventral Both, heterotopic (from dorsal to ventral or from ventral to dorsal) and homotopic grafting procedures displayed strong and weak migration. More cells were found to leave grafts inserted in distal positions (operation E) than in grafts in proximal positions. Furthermore, migration of ventral cells in the dorsal part seemed to be weaker than in the ventral part but note that the size of the graft was not standardized. Dorsally and ventrally migrating cells

97 Results originating from the same or the other part of the limb had in common that both took distal and proximal pathways in the same ratio (11 proximal – 12 distal) and did not cross the boundary towards ventral. Taken together, these series of experiments did not exhibit differences in migration between dorsal and ventral and were independent from the kind of grafting – heterotopic or homotopic.

3.4.2.7 Influences of origin, destination of the graft and the host-donor age relationship on migration

3.4.2.7.1 Origin and destination of the graft Grafts were collected from different proximal positions (origin) which influenced migration behavior. In one series, grafts were excised from proximal dorsal positions very close to the trunk and grafted homotopically very close to the trunk resulting in grafts that were detected completely within the trunk region after 48 hours of reincubation. Basically, no migration (6/7) was observed, even though quail donors were at least 1 HH stage older.

The destination of the graft affected migration behavior as well. Grafts collected from proximal positions (OP B and D) not too close to the trunk and inserted homotopically, the donor being 2-3 stages older than the host, revealed limited migration in 5/7 samples. In contrast, grafts cut out from the same position and grafted to distal locations (OP E) using the same charge of eggs with the same donor-host age-relationship, strong migration was observed, see section 3.4.2.3, in 7 out of 9 samples.

3.4.2.7.2 Host-donor age relationship This section analyzes the effect of stage differences between host and donor. Due properties of the incubator and the eggs, not all operations were performed with exactly two HH stages difference. In table 15, 9 out of 16 samples showing long-range migration were grafted from proximal positions to proximal positions (OP A, C, D). In 8 out of 9 of these samples, the donor quail was two or more stages older than the chick host. Only in one case long-range migration was observed in an operation with both, host and donor being at the same stage. In operation E, when proximal blocks were grafted into the distal tip of the wing, no difference was detected between samples operated at two or more HH stages difference (long range migration : n = 2:3) or with samples at less than 2 HH stages difference (3:4).

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3.4.3 Brief summary of grafting experiments

In all operations described in the last sections (3.4.2.2 and 3.4.2.3), cells migrated in distal and proximal directions and were never found to cross the boundary and migrate out of the part of the wing from where the graft was inserted. Migration behavior of cells in homotopic compared to heterotopic grafting procedures was similar. Distal insertion of ventral cells lead to dorsal and ventral migration at the same ratio, but no cells migrated within the central part of the wing. Mesenchyme blocks grafted into the central region within the chondrogenic zone did not exhibit migration if the graft had no access to mesenchyme. Segment overlapping muscle blastemas seemed to recruit cells from both, the ventral and dorsal part. Cells migrating proximally from grafts in the ventral proximal wing gathered at a specific position located caudally of the limb in the trunk close to the surface. The origin of grafts influenced the grafting behavior as well as the destination and the host-donor age relationship.

99 Discussion

4. Discussion

4.1 Analysis of LacZ reporter mice provides novel insight into the Lmx1b expression pattern Insertion of a LacZ reporter gene without disrupting endogenous Lmx1b expression turned the Lmx1b 3’LacZ mouse into an extremely interesting model to study the expression pattern in detail. Due to the properties of X-gal staining, specific methods of ß-galactosidase detection and the availability of modern tools like confocal microscopy, the distribution of Lmx1b expression could be analyzed even on a subcellular level. In comparison to other transgenic mice with reduced levels of transcription, the aim of this study was not to describe morphological alterations resulting from lack of gene expression. In order to form a basis for further investigations and to reveal new fields of expression, it was rather interesting in a first step to determine exactly where Lmx1b is expressed and in a second step to hypothesize and test interactions with other genes.

4.1.1 Lessons from Lmx1b genetics

Lmx1b is part of a transcriptional machinery regulating expression of other genes (German et al. 1992; Curtiss and Heilig 1998). It seemed possible that the transcription factor also affects its own expression. In the present study, it was shown that the level of ß-galactosidase staining and likewise the level of Lmx1b was two times stronger in homozygous Lmx1b 3’LacZ mice than in heterozygous animals in the limb. This finding indicated that Lmx1b expression was linear and increasing concentrations of the transcription factor did neither downregulate nor upregulate its own expression. This is in line with results from Riddle and coworkers. Retroviral misexpression of Lmx1 in chick did not induce endogenous Lmx1 expression and transcription was not positively autoregulated (Riddle et al. 1995).

Neither heterozygous nor homozygous Lmx1b 3’LacZ mice which were characterized in this study displayed obvious phenotypical alterations. Correspondingly, Lmx1b 3’LacZ/KO mice appeared normal. In these mice, one allele was partially deleted and the other one carried the complete Lmx1b gene in addition to the LacZ construct. These results suggest that one functional allele is sufficient in mice to exert effects of Lmx1b. Insertion of the

100 Discussion

LacZ construct might slightly affect Lmx1b expression but did not severely reduce the level of transcription. It is known that in heterozygous Lmx1b mutants, only mild changes of phenotype were observed in the eye (Pressman 2001). In contrast, haploinsufficiency in the human LMX1B locus leads to the Nail-Patella Syndrome (McIntosh et al. 1997; Dreyer et al. 1998). Possibly, human homozygous mutations are embryonic lethal, as this genotype has not been described in humans up to now. Differences in the degree of manifestation between humans and mice might be reflected by differential threshold requirements for Lmx1b activity (Chen and Johnson 1999). In all, it is suggested that Lmx1b does not autoregulate its own expression. Insertion of the LacZ reporter gene did not significantly alter endogenous Lmx1b expression. Animals with the genotypes LacZ/LacZ, LacZ/WT and LacZ/KO appeared normal. Thus, Lmx1b 3’LacZ mice are a valuable tool to study Lmx1b expression. Further understanding of Lmx1b may help to comprehend and finally treat orthopedic symptoms in patients with NPS.

4.1.2 Lmx1b detection in Lmx1b 3’LacZ reporter mice corresponds to known patterns and reveals novel sites of expression

One aim of this study was to analyze murine Lmx1b expression in all parts of the body except for neural tissue during all embryonic stages and in newborns. First expression of ß-galactosidase was detected by X-gal staining at E8.5 and in the outgrowing limb buds at E9.5, while earlier stages were devoid of staining. This finding is in line with reports from literature on murine Lmx1b expression (Cygan et al. 1997). On the contrary, first chick transcripts were detected at HH4 in the primitive streak (Yuan and Schoenwolf 1999). Differences between species might explain these findings, or furthermore, early expression in mice has not yet been a major point of interest and remains a field for future investigations. In the present study, Lmx1b expression was analyzed by means of ß-galactosidase detection. The X-gal reaction depends on a sufficient amount of ß-galactosidase. A slight delay from transcription to the production of a relevant amount of translated protein could be responsible for delayed detection of ß-galactosidase compared to Lmx1b RNA. However, this problem was not noticed at stages where data was available for direct comparison to in situ hybridization.

101 Discussion

I found that embryonic limbs always stained homogeneously without formation of gradients along the axes during all stages. Lmx1b expression was maintained in newborn limbs at high levels and present in adult animals. On the contrary, Dreyer and coworkers reported an anterior-posterior and a proximo-distal gradient of expression in dorsal tissues correlating with the degree of differentiation at E12.5 and at E16.5. Additionally, maximum expression was found at E14.5, finally disappearing in the limb until E18.5 (Dreyer et al. 2000). The different results might be explained by the different techniques employed for Lmx1b detection. X-gal staining is more sensitive than the in situ hybridization method Dreyer and coworkers used. Notably, even in adult human brain, dopaminergic neurons express LMX1B (Smidt et al. 2000), supporting the idea that Lmx1b expression is present in adults and required for normal function.

In the limb, Lmx1b expression was detected in cartilage, bones, tendons and connective tissue. Even chondrogenic zones were not excluded, which has been reported previously. However, the staining intensity was lower compared to surrounding tissues. These cell types and tissues are derivatives from the lateral plate mesoderm (Christ et al. 1977). These findings suggest that Lmx1b is expressed in all cells that develop from the lateral plate mesoderm. The mammary gland, teeth and hair follicles are derived from the ectoderm. This study revealed Lmx1b expression in the mammary gland and in the dermal papilla at the base of hair follicles. Expression during teeth development and the teeth phenotype in Lmx1b mutants were not examined. Further investigation in the role of Lmx1b in skin and hair development is an interesting future task. Interestingly, Msx2 mutants displayed similar defects in the mammary gland, the skull and other organs (Satokata et al. 2000) compared to mice targeted for Lmx1b (Chen et al. 1998a). Examination of liver, spleen and heart did not exhibit X-gal staining. As this staining method is sensitive and the control organs stained well in the same assay, it is suggested that these organs did not express Lmx1b. Nonetheless, novel expression was detected in the embryonic and adult lung. X-gal positive cells arranged in clusters of 5 to 10 cells in a row were located in the primary bronchioles. The function of these cells remains to be determined. A number of publications described expression of Lmx1 in chick in general (Parr et al. 1993; Riddle et al. 1995; Vogel et al. 1995; Giraldez 1998; Yuan and Schoenwolf 1999;

102 Discussion

Adams et al. 2000) and in the chick wing (Dealy et al. 1993; Parr et al. 1993; Riddle et al. 1995; Vogel et al. 1995). Only rare fields of murine Lmx1b expression have been examined in detail (Cygan et al. 1997; Chen et al. 1998a; Dreyer et al. 1998; Pressman et al. 2000; Smidt et al. 2000) and description in some organs lacked completely. Hence, this study provides novel findings on murine Lmx1b expression in the limb and in several organs.

4.1.2.1 Cells invading the limb are devoid of Lmx1b expression Lmx1b was shown to be expressed in the dorsal limb bud mesenchyme. It remained unclear whether all cells within the dorsal limb were Lmx1b positive or whether particular cell types did not express the gene. To address this issue, X-gal stained sections were examined. It was found that cells within the dorsal premuscle mass were devoid of Lmx1b expression. Confocal images of antibody stained limbs exhibited the same results. These findings suggest that myogenic cells do not express the gene. To confirm this result, double labeling with ß-gal and Lbx1 antibodies was performed. Lbx1 is a marker for myogenic precursor cells (Jagla et al. 1995). It was found that the entire population of Lbx1 positive myogenic precursor cells within the dorsal limb lacked Lmx1b expression. Up to now, Lmx1b has been characterized as a dorsal marker. This finding demonstrates that Lmx1b does not exclusively stain all dorsal mesenchymal cells. These results are supported by the finding that within the paraxial mesoderm and its derivatives, no Lmx1b staining was detected. Myogenic precursor cells originate from the somites (Christ et al. 1974b; Chevallier et al. 1977; Christ et al. 1977) and are therefore Lmx1b negative in the course of delamination from the lateral lip of the dermomyotome. It seemed possible that migrating cells initiate Lmx1b expression while entering the limb. However, this study showed that myogenic precursor cells remain Lmx1b negative. Moreover, it is possible that other cells invading the limb maintain their Lmx1b negative status. Cells and structures of the future limb are not only derived from the lateral plate mesoderm. Melanoblasts and Schwann cells are neural crest derived, endothelial and lymphatic cells originate from the somite and axons grow into the limb from the neural tube (Christ et al. 1977; Hollyday 1983; Solursh et al. 1987; Lance-Jones 1988; Beddington and Martin 1989; Serbedzija et al. 1989; Brand-Saberi et al. 1995; Christ and Ordahl 1995; Wilting et al. 1995). Histological examination of early mouse limbs displayed to a large extent myogenic precursor cells lacking Lmx1b expression. The major part of migrating cells consists of myogenic cells. In chick limbs it was shown that

103 Discussion

Schwann cells and axons do not appear in the limb before stage HH24 and melanocytes invade the limb after HH21 (Hollyday 1983; Serbedzija et al. 1989). All in all, it was shown that dorsal limb mesenchyme did not impose Lmx1b expression on myogenic precursor cells which maintained their Lmx1b non-expressing status. It can be assumed that additional cells invading the limb like melanoblasts, endothelial or lymphatic cells lack Lmx1b expression within the limb as well. Double labeling with specific antibodies for neural crest derivatives like HNK-1 (Rickmann et al. 1985; Bronner-Fraser 1986) or directed against endothelial cells like QH-1 (Pardanaud et al. 1987; Wilting et al. 1997) and Lmx1b could substantiate the absence of staining in these cell populations. Lmx1b appears to be an exclusive marker only for derivatives of the lateral plate mesoderm.

4.1.2.2 Lmx1b is likely to be involved in neuronal pathfinding in the limb Examination of limb sections showed that axons were devoid of X-gal staining, in contrast to the surrounding mesenchyme. This finding raises the question if neurons invading the limb express Lmx1b. In addition, it was shown that Lmx1b is expressed in the nuclei of cells. Lmx1b is also expressed in the neural tube. As the nuclei of limb motor neurons are located in the neural tube, it remained to be determined if motor neurons projecting into the limb express Lmx1b. Furthermore, this study showed that the motor nerve bifurcation at the base of the limb coincides with both, the proximo-distal and dorso-ventral boundaries. These findings are in agreement with results from Kania and coworkers (Kania et al. 2000). Motor and sensory neuron innervation are differentially regulated in the dorsal and ventral part of the limb bud and affected by presence or absence of Lmx1b (Chen and Johnson 1999). The lateral motor column (LMC) in the neural tube projects axons from a medial part into the ventral limb and from a lateral part into the dorsal limb (Landmesser 1978; Tosney and Landmesser 1985b, a; Tsuchida et al. 1994; Tosney et al. 1995). In contrast, in Lmx1b mutants, axons from the lateral LMC selected both, the ventral and dorsal trajectory (Kania et al. 2000).

The expression pattern described here suggests that the dorsal Lmx1b expressing mesenchyme could attract neurons growing from the lateral part of the LMC. In addition, it is possible that projections of the medial LMC are rejected by Lmx1b positive

104 Discussion mesenchyme, thus guiding the way into the ventral limb. Two other members of the LIM homeodomain family, Isl1 and Lim1 are differentially expressed in the LMC (Tsuchida et al. 1994). Thus LIM homeodomain proteins may play an important role in the pathfinding of limb axons by giving internal (Isl1, Lim1) and external (Lmx1b) guidance cues.

4.1.2.3 Differentiated muscle cell may lack Lmx1b expression The paraxial mesoderm and its derivatives, the somites, lacked Lmx1b expression. Myogenic precursors delaminate from the lateral lip of the somite (Christ et al. 1974b; Chevallier et al. 1977; Christ et al. 1977). Therefore, myogenic precursor cells are devoid of Lmx1b expression at the very beginning of migration. This study demonstrated that all Lbx1 positive myogenic precursors migrating within the dorsal premuscle mass did not initiate Lmx1b expression (see 4.1.2.1.). Furthermore, it was shown that Lmx1b expression was detected in areas of differentiated muscle cells. Different cell types are present within these muscle areas; the differentiated muscle cells themselves but also undifferentiated satellite cells and connective tissue. It was not excluded that some differentiated muscle cells turn on Lmx1b expression However, antibody staining on newborn muscle regions with MyoD and myogenin revealed many unstained nuclei, similar to X-gal staining. The level of staining in tissue surrounding the muscle was stronger. Given that myogenic precursor cells lack Lmx1b expression, induction of the gene is required by an unknown mechanism if differentiated muscle cells were positive. Only a subset of stained nuclei within muscle regions belongs to muscle fibers. The fact that other cell types are also present, suggests that only nuclei from connective tissue elements derived from the lateral plate mesoderm express Lmx1b. Differentiated muscle is assumed to be devoid of staining as it was shown for myogenic precursor cells. To confirm this hypothesis, double labeling with antibodies directed against differentiated muscle and Lmx1b could be performed.

4.1.3 Lmx1b is expressed during all stages of mammary gland development

To determine the relevance of Lmx1b expression, homozygous mutants were examined for morphological alterations in mammary glands. Severe defects during development resulted in a complete lack of the mammary glands at birth. These results indicate a basic requirement of Lmx1b for embryonic mammary gland development.

105 Discussion

As no data existed describing the expression pattern of Lmx1b in murine mammary glands, this study presents the first detailed examination throughout all embryonic and adult stages. The earliest expression was detected at E11.5 in the mammary gland primordium and maintained throughout embryonic development. After birth, expression was restricted to luminal epithelial cells, whereas myoepithelial cells and the stromal compartment were completely devoid of staining. This pattern persisted continuously without formation of gradients during all adult stages including pregnancy, lactation and weaning. These findings may in part explain reports of poor breast development in female human patients with NPS (Sweeney et al. 2003). The facts that (i) mutant mice lack mammary glands, (ii) the gene reveals a continuous and stable expression and (iii) human patients exhibit breast hypoplasia strongly indicate an important function of Lmx1b in the formation and maintenance of mammary gland morphology.

As breast development occurs to a large extent postnatally, Lmx1b gene transfer and overexpression in girls with NPS to avoid breast malformation are a potential future therapy. The high costs and side effects of retroviral application opposed to traditional cosmetic surgery must be taken into consideration. Up to now, interactions with other factors are completely unknown. Possible candidate genes include members of the WNT family (Weber-Hall et al. 1994; Brisken et al. 2000), homeobox containing genes (Friedmann and Daniel 1996; Phippard et al. 1996) and parathyroid hormone related proteins (Dunbar and Wysolmerski 1999). To gain insight into the nature of Lmx1b function during mammary gland development, the investigation into possible interacting molecules will be a challenging issue.

4.1.3.1 The relevance of Lmx1b for tumor development The present study revealed that Lmx1b expression was restricted to luminal epithelial cells in the mammary gland. Tumor cells are in more than 90% of the cases of luminal cell-like phenotype (Altmannsberger et al. 1986; Nagle et al. 1986; Dairkee et al. 1988). Consequently, Lmx1b may be involved in breast tumor development. In addition, Lmx1b acts on a transcriptional basis (German et al. 1992; Curtiss and Heilig 1998) and could therefore directly modify gene expression. Basic research on mammary glands is of great importance. About 185.000 cases of invasive breast cancer and 42.000 deaths occurred in the United States in the year 2000 (Lipman 2001).

106 Discussion

Common genes involved in breast cancer include the tumor suppressor genes BRCA-1, BRCA-2, p53 and erB2 (HER-2, neu), an epidermal growth factor receptor (Lipman 2001). Up to now, genes interacting with Lmx1b are completely unknown in the mammary gland. Lmx1b may play a role in tumorigenesis or its suppression. Thus, more investigations in the basic understanding of genetic interactions within the mammary gland are important to enhance preventive strategies and develop new therapies in the future. This study presents a new candidate, Lmx1b.

4.1.3.2 Extra-cellular matrix (ECM) and cell surface molecules are potential downstream targets of Lmx1b It is suggested that Lmx1b is involved in the regulation of extra-cellular molecules. Lmx1b mutants exhibited a strongly diminished expression of alpha chains of type IV collagen of the basement membrane in the kidney (Morello et al. 2001). In the eye, fibrillogenesis and the organization of the corneal stroma was perturbed, with absence of keratocan, a keratin sulfate proteoglycan (Pressman et al. 2000). Analysis of the spatial distribution of different ECM molecules in mammary glands revealed that collagen I, collagen IV and laminin are only expressed by stromal cells surrounding the ductal epithelium but not by epithelial cells (Keely et al. 1995; Woodward et al. 2001). Most studies in the mammary gland have been conducted on integrins and cadherins (reviewed by Hynes 1992; Knudsen et al. 1998; Hansen and Bissell 2000). Modification of cell surface and ECM molecules affects tumor progression via alteration of intracellular signaling (Schoenenberger et al. 1994; Sastry et al. 1996). In other words, the transcription factor Lmx1b as part of a transcriptional machinery, might directly or indirectly influence the regulation of the composition of the extra-cellular matrix in the mammary gland and in other organ systems. With regard to breast cancer, further insight into the effects of cell surface and ECM components could help to give more precise predictions and in a long term view, to inhibit tumor progression.

4.1.4 In the kidney, Lmx1b expression is restricted to podocytes and accounts for nephropathy

This study revealed low levels of Lmx1b expression in the mesonephros already as early as E11.5. Chen and coworkers described first expression of Lmx1b in the S-shaped body of the metanephric kidney at E13.5 (Chen et al. 1998a), and Morello and collegues in the

107 Discussion visceral epithelium of the glomerulum at E14.5 (Morello et al. 2001). The reason for weaker expression levels in the mesonephros detected in mice in comparison to other species could be due to the great variation in function between different animals. In mice, the mesonephros develops only rudimentary structures (Zamboni and Upadhyay 1981; Smith and Mackay 1991; Kaufman). Several publications have already described Lmx1b expression in the kidney (Chen et al. 1998a; Stark et al. 2000; Morello et al. 2001) but it has never been precisely determined which kind of cell type expresses Lmx1b. Therefore, double labeling with antibodies directed against ß-galactosidase (=Lmx1b) and WT-1 was performed. WT-1 expression is restricted to podocytes (Mundel et al. 1997; Pavenstadt 2000). The present study provides evidence that all WT-1 positive podocytes coexpress Lmx1b postnatally. These findings are in line with reports published after these experiments have been performed (Morello and Lee 2002; Quaggin 2002; Rohr et al. 2002). Lmx1b mutants still express WT-1, reasoning that Lmx1b is located downstream of WT-1 (Rohr et al. 2002) or on a parallel pathway.

Nephropathy is associated with the Nail-Patella syndrome and the first sign of renal involvement are usually proteinuria with or without hematuria (Gubler et al. 1980). The prognosis in patients ranges from spontaneous remission, progression to nephrotic syndrome or to lethal renal failure (Leahy 1966; Simila et al. 1970; Hoyer et al. 1972; Daniel et al. 1980). Proteinuria results from a degradation or lack of foot processes in podocytes, the glomerular phenotype of Lmx1b mutants (Miner et al. 2002; Rohr et al. 2002). Targeted disruption of Lmx1b in mice provided a very valuable model for further investigations in NPS-associated nephropathy. It has been shown in this study that renal Lmx1b expression is restricted to podocytes. This stands in line with the clinical findings of proteinuria and hematuria. Other kidney functions, exerted by other kidney cell types, like the metabolization of hormones and synthesis of erythropoietin, angiotensin II or cholecalciferol seem to be primarily unaltered.

108 Discussion

4.2 A new perspective of the dorso-ventral boundary 4.2.1 The razor-sharp line of expression at the boundary does not respect anatomical structures

This study showed that the boundary dividing the dorsal and ventral compartment in the limb was established at the very beginning of limb bud outgrowth at embryonic day 9. Lmx1b expression persisted throughout all embryonic stages in the dorsal limb bud mesenchyme, separated by a sharp boundary towards proximal and ventral. This razor-sharp division examined in single cell resolution raises basic questions on how such a clear division can be established at the very beginning of limb budding and furthermore, how it can be maintained even in areas of high proliferation. These simple but fundamental questions belong to the most intriguing ones in developmental biology. Up to day, they remain unresolved. This work takes part in the effort to describe and to finally understand the underlying mechanisms of boundary formation and maintenance. I analyzed the distribution of Lmx1b expression in dorso-ventral-segment overlapping structures at the boundary. Expression at the boundary seemed to follow an imaginary line between dorsal and ventral. Interestingly, it did not respect anatomical structures. Staining extended to the boundary and filled the part located in the dorsal compartment, even if it was just a small percentage. Therefore, it was impossible to assign expression to specific bones or tendons if positioned within both compartments. On the whole, Lmx1b was confirmed to be a marker for the dorsal part but not for individual structures.

4.2.2 Dorsal and ventral compartments are not maintained by repeling properties of all mesenchyme cells

When oil and water are added together and mixed, the hydrophobous and hydrophilous components separate quickly, forming a sharp line of division. Dorsal limb mesenchyme cells positive for Lmx1b exhibit a sharp boundary to ventral cells, similar to the oil and water experiment. To determine the sorting out behavior of limb cells, whole limb mesenchyme was dispersed and cultured in a micromass. This study showed that cells maintained their homogenous distribution. Primarily, this finding is astonishing. Even though the dorsal and ventral mesenchyme are divided by a sharp line of division, cells did not sort out. However, this result correlates with findings from Piedra and coworkers. They observed a persisting random pattern of Lmx1b expression in the proximal region of a

109 Discussion recombinant limb (Elisa Piedra et al. 2000). Inadequate culture conditions as a reason for non-sorting were ruled out as cultured cells were in good condition. Furthermore, cartilage islands had formed after 3 days of culture corresponding to description in literature (Ahrens et al. 1977; Maini and Solursh 1991). To evaluate the finding, the molecular basis needs to be considered. A number of classes of cell adhesion molecules are involved in the segregation of cells. Several mechanisms can lead to the separation of cells: (i) different cell types exhibit different types of adhesion molecules or (ii) different cell types have different amounts of cell adhesion molecules (Gilbert 2000). Cadherins belong to a group of calcium-dependent adhesion molecules which are anchored into the cells by a complex of proteins termed catenins. Cadherins have a crucial function in intercellular connections, spatial segregation of different cell types and in the patterning of structures and organs (Takeichi 1990). N-cadherin was shown to be expressed in a distinct pattern in the chick limb (Brand-Saberi et al. 1996a). Cell culture experiments demonstrated that sorting out of cells can be explained by different types and by different amounts of cadherins expressed on the cell surface (Takeichi et al. 1979; Nagafuchi et al. 1987). In conclusion, my findings suggest that Lmx1b does not directly or indirectly modify cell surface components of all dorsal mesenchyme cells. Therefore, mixed dorsal and ventral limb mesenchyme cells remain homogeneously distributed. However, it cannot be excluded that adhesion properties of a subpopulation of dorsal cells are modified by Lmx1b expression.

Up to now, it remains unclear how the maintenance of the sharp dorso-ventral boundary and the in vivo separation of the two compartments can be explained. Different model systems exist to analyze and explain in vitro pattern formation in micromass culture, the cell sorting model (reviewed by Steinberg 1996) and the reaction diffusion model (Turing 1952). In the cell sorting model, the separation of cells can be driven by dissimilar cell motility or differences in cell surface molecules or diverging properties of the extra-cellular matrix (ECM) leading to differential cell adhesion of two cell populations. Examples of the cell sorting out phenomenon are separately labeled proximal and distal limb cells. It has been shown in chick (Ide et al. 1994) and mice (Miura and Shiota 2000) that cultured cells form a nodule pattern after 24 hours. Depending on the kind of assay, cells in the limb reacted according to the sorting out model. However, limb cells positive and negative for Lmx1b

110 Discussion analyzed in this study did not. To explain this finding, a model established in a different species can be employed.

In Drosophila, a sorting out phenomenon of dissociated anterior and posterior cells was not observed (Fausto-Sterling and Hsieh 1987). This could be explained by the notion that only cells located close to the boundary possess a specific character necessary to separate the two compartments. The percentage of these cells in comparison to the amount of cells in the whole wing is small and therefore not sufficient to reveal separation of the two populations from anterior and posterior. There is only a minor chance that two cells with opposing characters would be located closely and reject each other. This might be also true for the finding in this study that all dorsal cells do not sort out from all ventral cells if differential adhesion persists only close to the boundary. Thus, differences on the cell surface or properties of the ECM between dorsal and ventral cells could not be assigned to Lmx1b expression with respect to the sorting out phenomenon. Nevertheless, it seems possible that a different, as yet undiscovered gene that is expressed solely close to the boundary is responsible for divergent cell adhesion, hereby leading to cell repulsion in dorsal and ventral cells. This could be evaluated by collecting only cells located in the center of the limb. In the Drosophila wing imaginal disc, several secreted proteins, Hedgehog (Hg), Wingless (Wg), members of the Wnt family and Decapentaplegic (Dpp) which belongs to the TGF-ß superfamily mediate the organization and patterning of the wing at the boundary (reviewed by Cohen 1993; Dahmann and Basler 1999). It is conceivable that cells in the central region of the vertebrate limb form a similar or different organizer region at the boundary. Some kind of yet unknown instructive mechanism exists as the straight line of separation between dorsal and ventral cells is maintained steadily and precisely, as shown in this study.

111 Discussion

4.2.3 Migrating cells respect the boundary between dorsal and ventral and do not change sides

Although it has been described which somites contribute to which individual muscle (Jacob et al. 1978; Beresford 1983; Zhi et al. 1996), the mere migratory pathways cells follow between somites and the base of the limb and within the limb remain widely unspecified in detail. This study addressed the issue of migration behavior in the chick limb. Blocks containing different migratory cells were collected from different proximal positions of the limb. The vast majority was represented by myogenic precursor cells. I demonstrated that in all grafting experiments designed to investigate the migratory pathways, migrating cells remained exclusively within the compartment they attained in the modus operandi. No cells were detected migrating from dorsal to ventral or from ventral to dorsal. These findings suggest that either migratory cells cannot cross the dorso-ventral boundary due to lack of permissive interactions. Alternatively, instructive signals were not given which attribute a dorsal or ventral destination to migrating cells. Howard Holtzer described the basic principles of instructive and permissive interactions (Holtzer 1968). Instructive interactions are involved to determine the state of a cell by initiating gene expression in the responder cell. Instructive tissue interactions influence the restriction- determination process and direct cells into a specific cell line. Permissive interactions do not affect the restriction-determination process but are required to allow normal development. The properties of the cells surface, the composition of the extra-cellular matrix or influences by hormones are examples for permissive interactions that allow expression of specific traits but do not impose a heritable condition of determination. Myogenic precursor cells express N-cadherin at high levels. The stationary mesenchyme along migratory routes is moderately positive while N-cadherin is absent in the central limb at the dorso-ventral boundary (Brand-Saberi et al. 1996a). Experimental modification of N-cadherin function in chick limbs suggests the requirement of N-cadherin for proper migration of myoblasts (Brand-Saberi et al. 1996a). Other molecules like fibronectin or hyaluronic acid are similarly distributed and were shown to affect migration (Toole et al. 1984; Jaffredo et al. 1988; Brand-Saberi et al. 1993). Taken together, the properties in the central part of the limb limit permissive interactions with myogenic precursor cells. This supports the finding that no migrating cells were detected in this area or were found to

112 Discussion cross the dorso-ventral boundary. Even if instructive interactions determined cells at the very beginning of migration, the composition of the central part might avert compliance to these signals. It was found in this study that in the proximal limb close to the trunk, migrating cells do not move between the two compartments. One reason to investigate in particular in this region was the appearance of the Lbx1 expression pattern. In situ hybridization for Lbx1 exhibited a ‘nose-like’ projection in the ventral proximal posterior limb pointed towards ventral. This was assumed to be a potential site of exchange. However, cells grafted into this region behaved in the same way as in other parts of the limb.

To be brief, grafting experiments indicated that cells remained either on the dorsal or on the ventral part of the limb and that no exchange occurred between the two compartments marked by Lmx1b expression. In addition, the assumption that Lmx1b expression is stable except for the distal part, gives a second hint that cells do not cross the boundary. Otherwise, they would be required to quickly change their expression status, going along with gradients of X-gal staining which were not observed at the boundary.

4.2.4 Different myogenic lineages in the dorsal and ventral premuscle mass

In this study, comparison of the dorsal to the ventral premuscle mass revealed interspecific differences and variations concerning different myogenic markers. In mice, Pax3 and Lbx1 positive areas in the dorsal and ventral premuscle masses exhibited the same size. Contrarily, in chick the ventral Lbx1 positive premuscle mass was wider compared to the dorsal Lbx1 positive premuscle mass or compared to the ventral Pax3 positive premuscle mass. These findings do not contradict the results from Gross and coworkers who showed that in an E10 mouse limb, myogenic precursor cells coexpress Lbx1 and Pax3. In mice, no differences were detected. Various explanations are conceivable to explain the presence of a larger ventral Lbx1 positive premuscle mass in chick as described. It is suggested that in chick, different subpopulations of myogenic precursor cells may be marked exclusively by either Pax3 or Lbx1. Pax3 might be downregulated earlier than Lbx1 in the shift from proliferation to differentiation, leaving more Lbx1 positive cells. Factors present in the ventral or dorsal limb may exert a stronger effect either on Lbx1 or on Pax3. The regulation of patterning in

113 Discussion the ventral premuscle mass might function in a different way than in the dorsal limb. Further investigation will be necessary to determine the nature of these interesting results. To address this issue, double labeling in chick with Pax3 and Lbx1 could be performed to reveal quantitative differences. Application of signaling molecules could provide insight in a possible regulation. Targeting of genes coexpressed in migrating myogenic precursor cells revealed major differences in the involvement of dorsal (extensor) and ventral (flexor) muscles. In Lbx1 mutants, the number of forelimb flexor muscles was reduced. All hindlimb muscles and forelimb extensor muscles were absent (Schafer and Braun 1999; Gross et al. 2000), while Mox2 mutants exhibited a lack of specific forelimb extensor and flexor muscles whereas all hindlimb muscles were present (Mankoo et al. 1999). Likewise, disruption of Met affected particular muscle groups (Maina et al. 2001). These reports from literature show completely different impacts of disruption of genes supposedly expressed in all myogenic precursor cells. In line with my findings, the differences in myogenic marker expression may be more distinct than currently assumed.

114 Discussion

4.3 Lmx1b expression with respect to the proximo-distal axis

The Apical Ectodermal Ridge (AER) which secretes FGFs, regulates the distal outgrowth of the limb. It interacts with other signaling centers and retains cells in the underlying Progress Zone (PZ) in a proliferative state (Saunders 1948; Summerbell 1974a; Niswander et al. 1993; Fallon et al. 1994; Martin 1998). The classical Progress Zone model proposed by Summerbell and coworkers (Summerbell et al. 1973) has been challenged by Dudley and coworkers (Dudley et al. 2002). It implies that progressive specification of fate is not only the result of the time cells spend in the PZ but fates are also established in a spatial scale. Along the proximo-distal axis, a gradient of differentiation exists with least differentiated, highly proliferative cells distally in the PZ and differentiating and specifying cells towards the base of the limb. After leaving the PZ, cells begin differentiation accompanied by condensations in chondrogenic zones and the formation of bony structures. The differences in differentiation certainly affect gene expression. I gave evidence that Lmx1b was expressed homogenously and not in a graded fashion along the proximo-distal axis in the limb. Nevertheless, distal Lmx1b expression responded differently to changes of gene expression or application of factors than proximal expression (Riddle et al. 1995; Vogel et al. 1995). This indicates a differential stability of Lmx1b expression along the proximo-distal axis which can not be derived from the expression pattern.

4.3.1 Proximal cells maintain their identity permanently

To determine if distal molecules can initiate Lmx1b expression, Wnt7a secreting cells and FGF8b recombinant protein were applied on ventral proximal mouse limb mesenchyme. I found that proximal ventral cells did not turn on Lmx1b expression. These results suggest that the combination of Wnt7a and FGF8b was not sufficient to trigger proximal Lmx1b expression. Alternatively, 3T3 cells did not secrete appropriate amounts of Wnt7a. Nevertheless, Wnt7a, a glycoprotein expressed and secreted by the dorsal ectoderm, is capable of Lmx1b induction in the ventral distal mesoderm. No induction was observed in the proximal limb and in the flank (Riddle et al. 1995). It remained unclear if the combination of factors including Wnt7a and other molecules present within the dorsal limb are sufficient to induce Lmx1b expression in ventral proximal cells. Therefore, different

115 Discussion sets of grafting experiments in different species were carried out in this study. Quail tissue blocks were grafted from the ventral proximal Lmx1 negative mesenchyme into various dorsal positions of a chick limb. I demonstrated that ventral proximal grafts maintained their non-expressing status in 100% of the cases. These findings indicate that factors present in the proximal dorsal limb are not sufficient to trigger Lmx1b expression in proximal ventral cells. On the other hand, these cells may have lost the competence to react to signals.

Different concepts underlie the process of morphogenesis and patterning. Competence is defined as the ability of cells to respond to a developmental signal (Waddington 1940). Tissue interaction at close range is termed proximate interaction or induction. At least two components are involved: a cell producing a signal, the ‘inducer’ and a cell which is being induced, the ‘responder’. Concerning my results, dorsal cells are the inducers and ventral proximal cells are the responders. Two types of inductive interactions have been described by Howard Holtzer (Holtzer 1968), instructive and permissive interactions (see section 4.2.3). Norman Wessels has proposed three principle characteristics of instructive interactions (Wessels 1977). (i) In the presence of tissue A, responding tissue B develops in a certain way. (ii) In the absence of tissue A, tissue B does not develop in that particular way. (iii) In the absence of tissue A, but in the presence of tissue C, tissue B does not develop in that way it does in (i). In the grafting experiments, the way would have been defined as the onset of Lmx1b expression and the adaptation of a dorsal fate. Ventral cells (A) did not respond to dorsal cells (B) raising the question if the prerequisite of competence was fulfilled.

To exclude the possibility that solely the combination of factors in the proximal limb and Wnt7a combined with FGF8b were not sufficient to trigger Lmx1b expression, proximal ventral mouse cells were grafted underneath the AER. I showed that Lmx1b expression was not initiated after 28 hours of culture using the sensitive X-gal detection system. These results showed that factors present within the dorsal limb were not sufficient to turn on Lmx1b expression in proximal ventral cells. These findings suggest a lack of competence to respond. This proximal stability of non-expression or expression has been described in numerous publications. Neither after ectoderm removal (Riddle et al. 1995; Araujo et al. 1998) and

116 Discussion

En1 overexpression (Logan et al. 1997) nor in mice targeted for Wnt7a (Loomis et al. 1998), proximal Lmx1b expression was affected. In contrast, in the distal limb ventral expression was ectopically induced in En1 mutants (Loomis et al. 1996) and after Wnt7a overexpression (Riddle et al. 1995). Distal dorsal Lmx1b expression was lost after ectoderm removal ((Riddle et al. 1995; Araujo et al. 1998), confirmed here) and En1 overexpression (Logan et al. 1997). All in all, a striking difference exists concerning the responsiveness of Lmx1b transcription to suppressive and inductive signals between proximal and distal parts of the limb. Seemingly, more mature proximal cells lost the competence to respond to Wnt7a, FGF8b and to other signals present in the proximal and in the distal limb as it was demonstrated in this study. These findings lead to the conclusion that at a ‘point of no return’ in limb development, cells “close” their genetic status concerning Lmx1b expression. They become incompetent to respond to signals and continue to maintain their expressing or non- expressing status permanently.

In a classical question posed by Spemann and coworkers, cells in grafted tissue blocks can either maintain their programmed fate (‘herkunftsgemäß’) or adopt a new fate by signals in the new environment (‘ortsgemäß’). Cells transplanted into the Msx1 expressing zone underneath the AER initiated Msx1 expression (previously known as Hox-7 (Robert et al. 1989)). Irrespective of its origin from expressing or non-expressing regions (Brown et al. 1993), these cells exhibited a position dependent (‘ortsgemäß’) behavior. In contrast, in my grafting experiments all proximal ventral cells behaved ‘herkunftsgemäß’ concerning Lmx1b expression.

4.3.2 The organizer region is not reestablished after dispersion of limb cells

Limb mesenchyme culture experiments provided further evidence for the stability of Lmx1b expression and the inability to reestablish an organizer region. I found that even single cells located at the margin of the micromass culture spot, maintained their expression. However, only slight influence or few factors from other cells are present in these marginal locations. These results support the idea of high stability of Lmx1b expression. Moreover, it was shown that distribution of cells remained random and did not reorganize after dispersion.

117 Discussion

The recombinant limb, described by Piedra and coworkers, could be considered as the in vivo version of micromass culture and exhibited similar results. The distal Lmx1b expression pattern which resembled the wild-type, diverged completely from the proximal random distribution of Lmx1b positive cells in the ventral and dorsal part (Elisa Piedra et al. 2000). All these results suggest that the proximal part is not capable of reestablishing an organizer region at the dorso-ventral boundary. Apparently, distal parts are not yet determined concerning Lmx1b expression.

4.3.3 Different migration behavior in proximal and distal limb regions

Results from interspecific grafting experiments carried out in this study, exhibited specific differences of the migration behavior with regard to the proximo-distal axis. Grafts collected from proximal positions very close to the trunk and transplanted homotopically, migrated only in few cases. In contrast, the same grafts inserted distally into the Progress Zone, migrated far in ventral and dorsal compartments. These findings suggest that different permissive interactions play a role in the proximal limb compared to the distal limb. In addition, the previously reported results of proximal and distal migration of grafts inserted into the Progress Zone were confirmed. Furthermore, less dependence of the donor-host age relationship than in proximal grafting procedures was corroborated (Brand-Saberi and Krenn 1991). The difference between strong migration of cells grafted distally and missing migration of grafts inserted proximally in this set of experiments, could be attributed to the gradient of differentiation of the surrounding host tissue. Proximal tissue of a limb is more differentiated (‘older’) than distal tissue at the same stage. Different factors exert their influence on the state of differentiation and on the adhesiveness of the stationary mesenchyme. These properties are part of permissive interactions and influence hereby migration behavior. FGFs secreted by the AER keep the distal mesenchyme in an undifferentiated state (Toole 1972). These factors were suggested to maintain distal cells in an uncommitted status concerning the proximo-distal identity (Niswander et al. 1993; Fallon et al. 1994) and possibly also with respect to the dorso-ventral axis (Riddle et al. 1995). Furthermore, members of the FGF family, present in the PZ, were implicated to promote myoblast proliferation (Edom-Vovard et al. 2001) and migration to the limb bud (Webb et al. 1997).

118 Discussion

Myogenic precursor cells express FGF receptor 1 (Olwin and Rapraeger 1992; Itoh et al. 1996) and presumably other subtypes. Therefore, it can be concluded that migration in the distal part of the limb is facilitated compared to proximal migration. Molecular influences by FGFs and potential other molecules present in the PZ participate in the preservation of the undifferentiated status and enhance migration. One candidate to increase the motility of myogenic precursor cells and maintain their undifferentiated state during migration is scatter factor/hepatocyte growth factor (SF/HGF) (Scaal et al. 1999). Heymann and coworkers did not observe migration towards beads soaked in SF/HGF. They concluded that SF/HGF does not act as a chemoattractant on myogenic precursor cells which has been proposed by Bladt and coworkers (Bladt et al. 1995; Heymann et al. 1996). These findings have been challenged by Scaal and collegues. They found an increase of migratory activity after SF/HGF application. In conclusion, a direct chemoattractive function was not attributed to SF/HGF. Nonetheless, it was suggested that SF/HGF was required to maintain motility of migratory cells (Scaal et al. 1999). Interestingly, the expression domain of SF/HGF moves from the proximal mesenchyme in younger limb buds to the distal mesenchyme at later stages. Myogenic precursor cells express c-met, the receptor of SF/HGF. The migratory pathway coincides with SF/HGF expression. Migration ceases in the limb at stage HH25 concurring to the latest stage of SF/HGF expression (Brand-Saberi and Christ 1992; Heymann et al. 1996; Scaal et al. 1999). Furthermore, SF/HGF may be the substrate to govern the underlying mechanisms described by the juvenility hypothesis (Brand 1987; Brand-Saberi et al. 1989; Brand-Saberi and Krenn 1991). To sum up, it is suggested that these permissive interactions exerted by SF/HGF, FGFs and other molecules can explain the strong distal migration of cells that move reluctantly in the proximal limb as described in this study.

In line with the idea that myogenic cells are not specified prior to migration (Schramm and Solursh 1990; Grim and Wachtler 1991) or during early invasion of the limb, it was shown in this study that migratory cells grafted into the PZ moved equally in dorsal and ventral routes. No cells were detected in the central part. It could be stated that molecular influences in the distal part re-specify myogenic cells with respect to their destination. However, if dorsal or ventral cells were grafted in proximal regions without presence of

119 Discussion distal influences, these cells behaved in the same way, independent of the origin. There was no indication to postulate a ‘homing’ mechanism implying that the grafted ventral cells seek to return to their original pathway. Still the guidance of myogenic cells remains obscure and no current model exists stating instructive interactions that explain the basic programming for pathfinding. Certainly, myogenic cells do not move at random within the limb, otherwise somites could not be specifically attributed to individual muscles. In all, myogenic precursor cell migration was shown to be different in proximal and distal regions which can be explained by different permissive interactions. Furthermore, it was indicated that myogenic precursor cells are not pre-specified concerning their dorsal or ventral destination. To investigate in the mechanism determining their destination will be a challenging future task.

4.3.4 Reverse proximal migration and myogenic attraction

It was shown that different parameters influence migration behavior, the donor-host age relationship, the origin or the destination of the graft. Additionally, the environment into which the graft was inserted had a positive or negative influence on migration intensity. I found that no migration occurred if grafts were transplanted into a chondrogenic zone. This result indicates that some kind of mechanism impedes migration. Indeed, it was reported that the properties of the extra-cellular matrix in chondrogenic zones like absence or low levels of hyaluronic acid, fibronectins or cadherins inhibit migration (Toole et al. 1984; Jaffredo et al. 1988; Brand-Saberi et al. 1993; Brand-Saberi et al. 1996a). I re-examined samples displaying only short-range migration. In cases when grafts were inserted close to a future muscle blastema, migrating cells rarely exhibited long-range migration but entered and remained in the area of the nearby located forming muscle. This finding indicates that muscle blastemas attract myogenic precursor cells. In addition, if the region of the forming muscle was located within the dorso-ventral boundary, it seemed that myogenic cells were recruited from the dorsal and the ventral migratory pathway. It is suggested that segment-overlapping muscles recruit cells from both compartments. In several different grafting procedures it was found that many cells from grafts inserted into the proximal limb migrated to proximal positions. In particular, cells moved to a part of the body wall located caudally of the ventral proximal limb and remained there. This location seems to be a major attraction point for migratory cells. Indeed, N-cadherin expression was described in this part of the body wall (Brand-Saberi et al. 1996a) and it is

120 Discussion therefore likely that proximal migration can occur. This was also shown by application of SF/HGF which resulted in reverse migration in the limb (Scaal et al. 1999). It is suggested that these cells in the body wall and in the trunk contribute to the formation of muscles of the rotator cuff or supplied pectoral muscles with myogenic material. Up to now, myogenic precursor cells originating from the lateral lip of the dermomyotome were mainly described with respect to muscles located within the limb. The finding that cells leaving grafts migrated in distal and proximal directions supports the idea that proximal migration is not an artefact or due to illegitimate orientation of myogenic cells. Proximal migration of myogenic precursor cells has been described before (Brand-Saberi et al. 1989; Brand-Saberi and Krenn 1991). Intriguingly, myogenic markers like Pax3 or Lbx1 are not present in this ventral location in the body wall at least not before HH25. The formation of proximal muscles and the attachment to the body wall occurs later. It remains unclear which cells supply myogenic material for limb muscles attaching to the body and how these muscles are formed. Origin and guidance of myogenic cells and the molecular mechanism for proximal limb muscle patterning leave many open questions for future research. This study suggests that after proliferation in the limb, reverse proximal migration triggered by attractants in the body wall are involved.

121 Discussion

4.4 Integration of different mechanistic models of limb axis formation with respect to Lmx1b expression

Figure D4.1: Dorso-ventral axis formation and differential stability of Lmx1b expression A) Regulation of Lmx1b expression The proximal and distal limb are regulated in a different way. En1, expressed in the ventral ectoderm suppresses ventral Wnt7a expression which leads to a ventral phenotype. In the distal dorsal limb, Wnt7a, expressed in the dorsal ectoderm, is required to maintain Lmx1b expression in the underlying mesenchyme. This results in a dorsal phenotype. It remains unclear whether a corresponding factor to Lmx1b in the ventral mesenchyme exists. Wnt7a is a short range signal. In En1 mutants, Lmx1b expression leaves a negative central part. The dotted line indicates that central Lmx1b induction may be mediated by other yet unknown factors affecting only the distal part or the whole limb. In the proximal limb, it remains to be determined which upstream factors induce Lmx1b expression. These signals could originate from the mesodermal compartment before outgrowth of the limb as suggested in this study. Alternatively, the ectoderm could signal directly to the underlying mesenchyme. B) Stability of the Lmx1b expression status Distal limb cells were shown to change their expressing and non-expressing status. In contrast, proximal cells are suggested to maintain their status permanently. This is thought to be the case in both, the dorsal and ventral compartment. NT, neural tube; PM, paraxial mesoderm; IM, intermediate mesoderm; LPM, lateral plate mesoderm

122 Discussion

The present study provided novel information to better understand the formation and maintenance of the dorso-ventral axis. The potential role of the transcription factor Lmx1b will be integrated in this concept. I showed that Lmx1b expression was present in the dorsal limb mesenchyme. It marked a sharp line of division towards the ventral limb. This bipartite pattern existed from the very beginning of limb outgrowth. It persisted at least throughout all stages of embryonic development and most probably throughout life. Interestingly, dispersed dorsal and ventral limb mesenchyme cells did not sort out from each other. Investigations in the stability of Lmx1b expression demonstrated that ventral proximal cells maintained their non-expressing status permanently. In contrast, in the limb mesenchyme I cultured, a distal loss of expression was observed. These findings have been discussed in previous sections. Here, they will be integrated in the current model of dorso-ventral axis formation.

In transgenic mouse models, the reversal of the patterning in the limb affects mainly the distal limb in En1, Wnt7a, Wnt7a/En1, Lmx1b/Wnt7a and Lmx1b/En1 mutants similar to ectopic ventral Lmx1b expression in En1 mutants and after Wnt7a or Lmx1b overexpression (Riddle et al. 1995; Vogel et al. 1995; Loomis et al. 1996; Cygan et al. 1997; Logan et al. 1997; Loomis et al. 1998; Chen and Johnson 2002). Only mice targeted for Lmx1b exhibit a complete ventralization of the limb (Chen et al. 1998a). It can be concluded that Lmx1b acts as mediator for the dorsal phenotype while the ventral pattern seems to be the default setting. In addition, these findings are in line with the findings in this study describing stable proximal and modifiable distal expression. I propose that the stability of Lmx1b expression differs completely in the proximal in contrast to the distal limb. Furthermore, I suggest that ventral limb cells behave in the same way regarding the maintenance of their non-expressing status: very stable proximal (as shown by the grafting experiments) and unstable distal (as shown by loss of expression after ectoderm removal). The gene itself or its regulatory complexes may be modified in an unknown way to prevent transcription (dorsal - on, ventral - off) from being altered.

According to the current model of distal dorso-ventral patterning (Johnson and Tabin 1997; Chen and Johnson 1999, 2002), En1 is expressed in the ventral ectoderm where it suppresses ventral Wnt7a expression. Wnt7a, expressed in the dorsal ectoderm, induces Lmx1b expression in the dorsal mesenchyme. Mechanisms to control proximal Lmx1b

123 Discussion expression are completely unknown. Besides, these genes have been proposed to play a role in anterior-posterior patterning (reviewed by Chen and Johnson 2002). Evidence was given that not only factors present within the limb bud account for the establishment of the limb axes. Already in 1971, it was shown that somites contribute to limb axes formation (Kieny et al. 1971). Likewise, Michaud and coworkers produced a bidorsal limb when the limb region was flanked by two rows of somites or when a filter was placed lateral to the presumptive limb region (Michaud et al. 1997). These results indicate that somites produce a dorsalizing factor and that the lateral somatopleure produces a ventralizing signal. In addition, fate mapping of the limb ectoderm revealed that the ectoderm overlying the somites and the intermediate mesoderm gives rise to the future dorsal limb ectoderm. The ectoderm covering the lateral somatopleure generates the future ventral limb ectoderm and ectoderm overlying the presumptive wing develops only into the AER (Altabef et al. 1997; Michaud et al. 1997). Ectoderm – mesoderm recombination experiments by Geduspan and MacCabe lead to the conclusion that ectoderm is not specified before stage HH14 (Geduspan and MacCabe 1987, 1989). The model for the establishment of the dorso-ventral axis by Michaud and coworkers suggested that the paraxial and the intermediate mesoderm signal either directly to the presumptive limb mesoderm or to the overlying ectoderm. This ectoderm will give rise to the future dorsal limb ectoderm. In a second step, it instructs the underlying dorsal limb mesoderm (Michaud et al. 1997). The ventral compartment of the limb would be specified accordingly by the lateral somatopleure. In this study it was shown that the bipartite expression pattern of Lmx1b exists from the very beginning of limb outgrowth. The idea that the limb mesoderm may be instructed directly from the adjacent mesoderm supports this finding. The onset of Wnt7a and Lmx1b expression in the limb at stage HH15 correspond to each other (Riddle et al. 1995). Lmx1b expression is restricted from the very beginning of limb budding to the dorsal part and extends to the center of the limb. In order to establish and maintain a sharp boundary between dorsal and ventral, a signaling center must have been established. It seems improbable that short range factors from the dorsal ectoderm alone can exert these functions. However, it is not excluded that the signal from the dorsal ectoderm is mediated by other factors. More likely, the limb mesoderm is instructed directly by the paraxial, intermediate mesoderm for its dorsal aspect and from the lateral

124 Discussion somatopleure for its ventral aspect. Besides, signals from the ectoderm would most probably establish an asymmetrical expression pattern at the very beginning. Thus, the importance of Wnt7a and En1 expression would not be reflected in the establishment of the dorso-ventral polarity but in its maintenance, particularly in the distal limb. If an organizer exists in the vertebrate limb, it is suggested that this organizer and Lmx1b expression are established by signals from the adjacent mesoderm.

Interestingly, ectopic expression of Lmx1b in the distal ventral mesenchyme in En1 mutants covered only a surface-close region leaving a broad Lmx1b negative field between the dorso-ventral boundary and ectopic ventral expression (Cygan et al. 1997). It is assumed that WNT signaling operates only over a few cell diameters (Gonzalez et al. 1991; Nusse and Varmus 1992), which could account for the restricted surface-close distal expression. In contrast to En1 mutants, retroviral overexpression affected the whole limb mesenchyme and induced ventral Lmx1b expression from surface to the center of the limb (Riddle et al. 1995). However, it remains unclear if a secondary mechanism is necessary to induce and maintain distal dorsal Lmx1b expression in the central part, as WNT signaling from the ectoderm obviously does not act that far. Furthermore, these findings support the idea that Wnt7a which initiates expression parallel to Lmx1 before outgrowth of the limb is not sufficient to control Lmx1 expression including the establishment of the dorso-ventral axis.

Taken together, it was illustrated that dorso-ventral limb patterning can only be explained in a complex model in which Wnt7a might play a less important role than proposed after its discovery and analysis 5 to 10 years ago. The major importance of Lmx1b for the configuration of the dorsal patterning may be taken as key tenet. Its expression was suggested to be induced from the adjacent mesoderm. It was indicated that proximal cells are determined in terms of stability of the Lmx1b expression status. In the future, the discovery of the regulation mechanism in the proximal limb and a genetic approach to uncover factors upstream of Lmx1b and to reveal its regulatory elements should further clarify the function and position of the gene in the developmental network.

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165 Tables and Figures

6. Appendix 6.1 Tables and Figures

Table 1: Overview of crosses set up during the present study...... 32 Table 2: Overview of interspecific grafting procedures ...... 37 Table 3: Assays for linearization of Lbx, Lmx1 and Pax3...... 50 Table 4: In vitro transciption assay...... 51 Table 5: Probes for Southern blotting...... 52 Table 6: Enzymes, buffers and fragments in Southern genotyping...... 52 Table 7: Primer sequences for LacZ PCR ...... 54 Table 8: Cycle settings for LacZ PCR...... 54 Table 9: Primer sequences for X/Y PCR...... 55 Table 10: Cycle settings for X/Y PCR ...... 55 Table 11: Embryo and tissue fixation times for X-gal staining...... 56 Table 12: Equilibration time in different tissues and embryonic stages...... 57 Table 13: Fixation times for frozen section...... 58 Table 14: Overview Wnt7a and FGF-8b application on cultured mouse limbs...... 89 Table 15: Overview of interspecific grafting experiments...... 92

Figure I1.1: Phylogenetic tree illustrating the relationship of LIM homeodomain family members...... 4 Figure I1.2: Expression of Lmx1, Wnt7a and En1 in the wild-type limb...... 9 Figure I1.3: Chick embryos after 50 and 72 hours of reincubation...... 12 Figure I1.4: Scanning electron micrographs of chick embryos incubated for 50 and 72 hours...... 13 Figure I1.5: Skeletal pattern of the chick wing and the three major axes ...... 13 Figure I1.6: Expression of Sonic Hedgehog, FGFs and Wnt7a in the limb...... 15 Figure I1.7: Expression in the Drosophila wing disc ...... 18 Figure I1.8: Maintenance of a compartment boundary ...... 19 Figure I1.9: Schematic of myogenic precursor cell migration...... 20 Figure M2.1: Construct of Lmx1b 3’LacZ (A) and Lmx1b KO mice (B) and the wildtype locus...... 31 Figure M2.2: Lmx1b 3’LacZ mouse embryo at stage E15.5 stained with X-gal...... 33 Figure M2.3: OP A - ventral to dorsal...... 38 Figure M2.4: OP B – dorsal to ventral ...... 38 Figure M2.5: OP C – ventral to ventral ...... 38 Figure M2.6: OP D – dorsal to dorsal ...... 38 Figure M2.7: OP E – ventral into the Progress Zone ...... 39 Figure M2.8: OP B – dorsal to ventral ...... 39 Figure M2.9: Filtration apparatus...... 44 Figure M2.10: X-gal molecule: 5-bromo-4-chloro-3-indolyl-ß-D-galactoside...... 56 Figure M2.11: Illustration of double labeling with antibodies from the same species...... 62 Figure D4.1: Dorso-ventral axis formation and differential stability of Lmx1b expression...... 121

166 Abbreviations

6.2 Abbreviations AER Apical Ectodermal Ridge BABB benzyl alcohol, benzyl benzoate bHLH basic helix-loop-helix BM basement membrane BMP Bone Morphogenetic Protein bps base pairs C. elegans Caenorhabditis elegans DNA deoxyribonucleic acid dpc dies post coitum ECM extra-cellular matrix Ex embryonic day x FGF Fibroblast Growth Factor GBM glomerula basement membrane H&E hematoxylin and eosin HH Hamburger and Hamilton (1951) IM intermediate mesoderm KO knock out LCM lateral motor column LPM lateral plate mesoderm NPS Nail-Patella Syndrome OD optic density PCR polymerase chain reaction Px postnatal day x PZ Progress Zone RNA ribonucleic acid Shh Sonic Hedgehog ß-gal ß-galactosidase TEB terminal end bud WT wild-type WT-1 Wilms’ tumor gene X-gal 5-bromo-4-chloro-3-indolyl-ß-D-galactoside ZPA Zone of Polarizing Activity

167 Acknowledgements

6.3 Acknowledgements I am indebted to gratitude to Prof. Dr. Beate Brand-Saberi for arranging my research stay in Houston, Texas, U.S.A. Her constant support while I was in Freiburg and abroad gave me the feeling of always having a contact person for small organizational and intriguing scientific questions. She was engagé in supplying me with information. I am very thankful for making it possible to participate in and contribute to inspiring meetings and for very valuable advice on this manuscript even while being out of Freiburg.

I am indebted to gratitude to Prof. Dr. Randy Johnson for giving me the opportunity to work in his laboratory. I am very grateful for his very friendly welcome in Houston and quick integration into his laboratory where he introduced me to scientific work and guided my scientific thinking at the bench and on the whiteboard. He stimulated my curiosity, rendering science an even more fascinating field to work in, and also reminded me that there is a world outside the laboratory. The year I spent in Houston was an inestimably precious experience of life.

I am very grateful to all laboratory staff members of Prof. Dr. Beate Brand-Saberi, Prof. Dr. Randy Johnson, and Prof. Dr. Bodo Christ for helping me to answer simple and delicate questions in every-day work. I gratefully acknowledge the excellent technical assistance and the stimulating discussions in a very friendly atmosphere. I really enjoyed working with them all. I am deeply gratified to Verena Dathe who taught me experimental techniques during my first rotation in the laboratory and made me curious to experience more basic research and in particular, developmental biology.

I also would like to thank many researchers from other laboratories in MD Anderson Cancer Center, in Baylor College of Medicine and in the Institute of Biology in Freiburg for valuable technical advice and interesting discussions.

I owe endless thanks to my wonderful father for supporting and encouraging me at all times and to my precious friends for always being there when I was in need to share my dreams and fears and for valuable feedback.

Thank you!

168 Curriculum vitae

6.4 Curriculum vitae Personal data Name: Heiko Schweizer Date of Birth: August 10th, 1976 Place of Birth: Freiburg im Breisgau, Germany

School and Academic Career 1983 - 1987 “Sonnenrain“ Elementary School in Radolfzell, Germany 1987 - 1996 “Friedrich-Hecker“ Grammar School in Radolfzell, Germany 1996 Final exam “Abitur“ (Overall mark: 1.3) 1996 - 1997 Rescue Service (German Red Cross) in Radolfzell, Germany October 1997 Begin of Medical studies at the Albert-Ludwigs-University in Freiburg im Breisgau, Germany 1999 Preclinical examination (Overall mark 3.0) 2000 First Medical State Examination (Overall mark 2.0)

Practical Experience August 1996 Clinical Clerkship in the Department of Anaesthetics and in the ICU in the General Hospital Radolfzell, Germany September 1997 Nurse practical in the Department of Internal Medicine in the General Hospital Radolfzell, Germany August / Nurse practical in the Department of Internal Medicine and in the September 1998 Department of Anaesthetics in the General Hospital February / Clinical Clerkship in the Department of Internal Medicine March 2000 (Prof. Dr. Kley) at the Hegau General Hospital Singen, Germany August 2002 Clinical Clerkship in the family practice of Dr. P. Merk in Freiburg- Munzingen, Germany September 2002 Clinical Clerkship in the Department of Ophthalmology (Prof. Dr. Dr. Hartmann), Virchow-Hospital, Charité, Berlin, Germany March 2003 Clinical Clerkship in the Department of Neurology (Prof. Mancardi), Ospedale S. Martino di Genova, Italy April 2003 Clinical Clerkship in the Department of Urology (Prof. Dr. Wetterauer), University Hospital Freiburg, Germany

169 Curriculum vitae

Scientific Career December 1999 Start of experimental research work in the laboratory of Prof. B. Brand-Saberi at the Institute of Anatomy and Cell Biology at the Albert-Ludwigs-University Freiburg, Germany 2000-2001 Continuation of experimental research work in the laboratory of R.L. Johnson, Ph.D in the Department of Biochemistry and Molecular Biology, MD Anderson Cancer Center; Houston, Texas, USA 2000-2001 Classes in “Developmental Biology“ at Baylor College of Medicine, Houston, Texas, USA 2001- Continuation of experimental research and Medical studies at the Albert-Ludwigs-University Freiburg, Germany 2001/2002 Tutor for second year medical students in anatomical dissection classes at the Institute of Anatomy in Freiburg, Germany

Publications, Abstracts and Poster presentations Schweizer H, Johnson RL, Brand-Saberi B. (2004) Characterization of migration behavior of myogenic precursor cells in the limb bud with respect to Lmx1b expression. Anat Embryol 2004 Apr; 208(1):7-18; accepted 21 Nov 2003

Schweizer H, Johnson RL, Brand-Saberi B (2003) Characterization of migration behavior of myogenic precursor cells in the limb bud with respect to Lmx1b expression. Molecular Biology of Muscle Development and Regeneration, Banff, Canada

Schweizer H, Johnson RL, Brand-Saberi B (2002) Migration behaviour of myogenic precursor cells in the limb bud with respect to dorso-ventral patterning. Meeting of the Anatomical Society of Great Britain and Ireland „How to make a hand“, Dundee, Scotland

Schweizer H, Johnson RL, Brand-Saberi B (2002) Migration behaviour of myogenic precursor cells in the limb bud with respect to dorso-ventral patterning. Meeting of the Deutsche Anatomische Gesellschaft, Würzburg, Germany

Schweizer H, Brand-Saberi B, Johnson RL (2001) Analysis of C-Lmx1 expression and cell migration in the limb bud of quail-chick chimeras. Genes & Development Retreat on Mustang Island, Texas, USA

170 Declaration

zum Antrag auf Zulassung zur Promotion zum Dr. med.

Heiko Schweizer

6.5 Declaration (Erklärung über die Beteiligung Dritter)

Erklärung über Beteiligung Dritter

Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.

...... (Datum) (Unterschrift)

171Figures BFigures

Tableofcontents ad3.1.1GenotypingbySouthernblottingandPCR...... B1 ad3.1.1GenotypingandsexdeterminationofLmx1b transgenicmice...... B2 ad3.1.2ExpressionlevelofheterozygousandhomozygousLmx1b3'LacZ mice

andlocalizationofß-galactosidase...... B3 ad3.2.1WholemountX-galstainingofLmx1b3'LacZ mice...... B4 ad3.2.1.1Lmx1b expressionduringlimbdevelopmentonsections...... B5 ad3.2.1.2Detailsofnewbornlimbexpression...... B6 ad3.2.1.3Lmx1b marksasharpboundarybetweendorsalandventral...... B7 ad3.2.1.5Lmx1bandLbx1 arenotcoexpressedinthedorsallimb...... B8 ad3.2.2.1Embryonicmammaryglanddevelopment...... B9 ad3.2.2.5K14,K18andLmx1bantibodystaining...... B9 ad3.2.2Wholemountmammaryglands...... B10 ad3.2.2Mammarygland:Detailsofwholemounts...... B11 ad3.2.2.3MammaryglandsectionsI:P34topregnancy...... B12 ad3.2.2.4MammaryglandsectionsII:Lactationtoweaning...... B13 ad3.2.3Lmx1b expressioninthekidney...... B14 ad3.2.4.2.1Lmx1b expressioninadultandembryonicorgansI...... B15 ad3.2.4.2.2Lmx1b expressioninadultandembryonicorgansII...... B16 ad3.2.4.3WholemountLmx1bexpressioninaP26LacZ/LacZ mouse...... B17 ad3.3.1MicromasscultureofLmx1b3'LacZ limbmesenchyme...... B18 ad3.3.2Graftingandcultureoflimbmesenchymefromtransgenicmice...... B19 ad3.3.3EffectsofWnt7aandFGF8bapplicationonmouselimbs...... B19 ad3.3.4Inductionofventralproximallimbmesenchyme...... B20 ad3.4.1Lbx1 expressioninthequailandchickwing...... B21 ad3.4.2Interspecificgraftingexperiments-tableI...... B22 ad3.4.2Interspecificgraftingexperiments-tableII...... B23 ad3.4.2Interspecificgraftingexperiments-tableIII...... B24 B1Figures 3.1.1GenotypingbySouthernblottingandPCR

11.1Kbps(Wild-type)

7.4Kbps(LacZ)

WT-/-+/-

Figure1.1:GenotypingofLmx1b3’LacZ micebySouthernblotting. GenomicDNAfromatailpreparationwascutwithBamHI.Wild-typeanimalsdisplayedonebandat11.1Kbps andmutantsoneat7.4Kbpsafterhybridizationwitha700bpsfragmentfromthe47BIconstruct.

800bps(+/-and-/-)

Figure1.2:GenotypingofLmx1b3’LacZ micebyPCR PrimersweredirectedagainsttheLacZinsertinLmx1b3’LacZ animals.Both,heterozygousandhomozygous miceexhibitedamplifiedproductsof800bpsoflengthwhereasinwild-typeanimalsthisbandwasabsent. B2Figures 3.1.1GenotypingandsexdeterminationofLmx1b transgenicmice

A 11.1Kbps (Wild-type) 7.4Kbps (LacZ)

12345 LacZ:+++++ Deletion:++-+- Wild-type:--+-+

B 12Kbps (Deletion)

4.3Kbps (Wild-type)

Figure1.3:GenotypingofLmx1b3’LacZ/LacZmicecrossedwithLmx1bKO+/- mice bySouthernblotting. GenomicDNAwascutwithBamHIandprocessedfortwoSouthernblotsusingtheprobe47BItodetectaLacZ insertinpanelABandtheprobe47B2tovisualizeadeletioninpanel. A)Wild-typeanimalsshowedonebandat11.1KbpsandLacZpositiveanimalsonebandat7.4Kbps. B)Wild-typeorLacZinsertsdisplayedonebandat4.3Kbpsandmutantsoneat12Kbps. Animalsinlanes1,2and4carriedthegenotypeLmx1bLacZ/deletion andanimalsinlanes3and5were characterizedby.Lmx1bLacZ/wild-type

300bps(X) 280bps(Y)

XXXXXYXYXY

Figure1.4:Sexdeterminationbypolymerasechainreactionofyolksacpreparations fromLmx1b3’LacZ embryos. PrimersrecognizingsequencesonXandYchromosomeswereusedtodeterminethesexofembryos.Female animalsshowedonlyonebandat300bps(X)whereasmaleanimalsdisplayedtwobands,oneat300bps(X)and oneat280bps(Y). B3Figures 3.1.2Expressionlevelofheterozygousandhomozygous Lmx1b3’LacZ miceandlocalizationofß-galactosidase

A B

LacZ/LacZ LacZ/WT

Figure1.5:Expressionofß-galistwotimesstrongerinLmx1b3’LacZ/LacZ thanin Lmx1b3’LacZ/WT mice. SectionsoflimbsatstageE10.5ofLmx1b3’LacZ/LacZ(AB)andLmx1b3’LacZ/WT ()micewerestainedwitha polyclonalIgGantibodydirectedagainstß-galactosidase,theproductoftheLacZgene.Assecondaryantibody, Alexa488wasemployed(greenfluorescence)andnuclearcounterstainingwasperformedwithDapi(blue). Bothsectionswereprocessedinthesameassayunderidenticalconditionsandphotographsweretakenwiththe sameexposuretimestoallowdirectcomparisonofthestaining.Expressionlevelsofß-galactosidasewerehigh ingeneralandanimalshomozygousfortheLacZ genewerestainedtwotimesstronger(A)thanheterozygous animals(B).

C D

E

Figure1.6:InLmx1b3’LacZ mice,ß-galactosidaseislocatedinthenucleusofthecell. Asectionfromadorsalpartofalimbwaslabeledwithaß-galactosidaseprimaryantibodyandanAlexa488 secondaryantibody(CD-green)andcounterstainedwithTOPRO-3(-blue),whichstainedonlythenucleiof cells.Twochannelconfocalmicroscopeimagesweresplitandrecombinedin(.E) ß-galactosidaseandTOPRO-3nuclearstainingwere100%colocalizedimplyingthattheLacZexpression productwasonlylocatedinthenucleiofcells.Noteaswellthatnotallcellsinthedorsalpartofthelimbwere positiveforß-galactosidase. B4Figures 3.2.1WholemountX-galstainingofLmx1b3’LacZ mice

A B C

D E F

G H I

J

Figure2.1:OverviewofX-galstainedembryosfromE7.5toE14.5. Lmx1bexpressionintransgenicmicecarryingaLacZ reportergene.Noovertphenotypewasfound.Incontrast toearlyLmx1 -RNAdetectioninchickembryos,atE7.5(A)nostainingwasfound.ThefirstX-galreactionwas detectedatE8.5(BC)intheneuralfoldsanddevelopingbrain.AtE9.5(),whenthelimbbudstartedtogrowout, Lmx1b expressionwasalreadyrestrictedtothedorsalmesenchymeformingasharpboundarytoventral(seehigh magnificationin(JD))andtoproximalregions.Thispatternpersistedthroughoutallembryonicstages.()E10.5; (EFG)E11.5;()E12.5;()E13.5;(HI)E14.5;highmagnificationofalimbatE13.5in(). B5Figures 3.2.1.1Lmx1b expressionduringlimbdevelopmentonsections

A E10.5 B E10.5C E10.5

D E11.5E E11.5F E13.5

G E13.5H Newborn

I E13.5J E15.5K E15.5L E15.5 *

M E9.5N E15.5 O E17.5P E17.5 *

Figure2.2:Lmx1b stainedmesenchyme,tendons,chondrogeniczonesandconnectivetissue. Lmx1b3'LacZ embryosweresectionedatdifferenttimepointsofdevelopment,stainedwithX-gal,eosin(A,C- P)andhematoxylin(G,H,NB)orwithß-galantibodies()andDapi.Lmx1b wasexpressedinthedorsallimb mesenchyme,theneuraltubeandtheoverlyingectodermbutnotintheparaxialmesoderm(E9.5in(M),E10.5in (AD)andE11.5in()).Inthelimb,theseparationofthedorsalLmx1b expressingmesenchymefromtheventral non-expressingdomainisclearlyvisibleuntilstageE13.5(F,G)persistingduringfurtherdifferentiationinto varioustissues(E15.5in(J),E17.5in(O),transversalsectionatE15.5in(LP),atE17.5in(), aswellasin newbornlimbsin(HI)andFig.2.3).In(),chondrogeniczones(asterisk),muscleregions(arrowhead)and tendons(arrow)inthedorsalpartofanE13.5limbwereLmx1b positive.Somemuscleareaswerecompletely devoidofstainingatE15.5(starin(NK)).Figure()displaysthegleno-humeraljointatE15.5,stronglystained tendonsandpartiallystainedskeletalparts.Inolderembryos,thelocationoftheboundarycorrespondedtoan 'imaginaryline'betweendorsalandventral(J,H). B6Figures 3.2.1.2Detailsofnewbornlimbexpression

A B C

D E F *

G H I

J K L

M N O

Figure2.3:Innewbornlimbs,Lmx1b expressionremainsrestrictedtodorsaltissues. Theoverviewofadistallimbin(A)showsthedorsalrestrictionofLmx1b expressionformingasharpboundary (C,DE)alsowithinstructureslikejoints().Mostofthecellsandstructuresinasectionthroughdorsaldistal limbs(B)werestained.Noteexpressionunderneaththenailbodyin(C).Tendons(arrowin(FK),())and connectivetissue(arrowheadin(F))werestronglystainedwhereascolorproductswereabsentinaxons(asterisk in(F)).Sectionsofmuscleareasin(G,H,I,J,L)demonstratedthatconnectivetissuecomponentsseparatingthe musclefiberswerepositiveforLmx1b whilemanynucleiinmuscleregionsremainedunstained.Inhairfollicles (M),expressionisrestrictedtothedermalpapilla(N,O)inthebase.AllsectionswerestainedwithX-galand eosinand(I,L,O)werecounterstainedwithhematoxylin. B7Figures 3.2.1.3Lmx1b marksasharpboundarybetweendorsalandventral

A E11.5B E11.5 C E11.5D E11.5

E E13.5F E13.5G E13.5H E13.5

I E11.0J E10.5 K E13.5L E14.5

M E15.5N E13.5 O E15.5P E13.5

Figure2.4:Analysisonsinglecellresolutionconfirmedtheexistenceofasharpboundary. LimbsfromLmx1b3'LacZ transgenicmiceweresectionedalongthedorso-ventral(A,B,C,E)andanterior- posterioraxis(D,F,G,HO)atdifferentstages,stainedwithX-galandeosin(except).Lmx1b didnotonlymarka boundaryfromdorsaltoventral(A-H,J-L,arrowheadinC),butalsofromproximaltodistal(I,JC,arrowin). Evenonsinglecellresolution(BO),theboundarywasverysharp(confirmedbyß-galantibodystainingin()and persistedatlaterstages(E13.5showninF-H,N,P)fromproximal(F)todistal(G,H). AnalysisofthedistributionofLmx1b expressioninmuscleregionsin(M)atE15.5demonstratedthatwithinone musclesharpboundarieswereformed.Onlyconnectivetissueelementsbutnomusclecellsseemedtobe positive.In(NP),thechondrogeniczonewasdevoidofstainingwhereasin()halfoftheformingbonewas stained.(F)showsatransversesectionofaE13.5zeugopod.Withinthedorsalpartmanyunstainedcellswere found,aswellasin()C . B8Figures 3.2.1.5Lmx1bandLbx1 arenotcoexpressedinthedorsallimb

A B Lbx1

C Lmx1b

D DAPI

E E10.5F E11.5G E11.5

H E10.5I E13.5J HH25 K HH24

L TOPRO/Lmx1bM NB N NBMyoDO NBMyogenin

P E10.5 Q E11.5 R E11.5S E11.5

Figure2.5:MyogenicprecursorcellsdonotcoexpressLmx1b inthedorsalpartofthelimb. X-galstaining(H,ML)andantibodystaining()onsectionsofthelimbshowedcellsdevoidofstaininginthe dorsalpart,andmorespecific,withinthepremuscleregion(I).Doublelabelingwithantibodiesdirectedagainst ß-gal(=Lmx1b)andLbx1,amarkerformyogenicprecursorcells,wasperformedonE10.5(E)andE11.5(A-D, F,G)mouselimbs.NoneoftheLbx1positivemyogenicprecursorcellswascolabeledwithLmx1bsupporting theideathatcellsmigratingintothelimbbuddidnotturnonLmx1b expression.Othercelltypesmayalsobe negativeforLmx1b asitcanbederivedfromimagesofthedorsalpart(P-S)showing axonsinvadingthelimband singleunstainedcells.WholemountinsituhybridizationonchickembryosatstagesHH24andHH25()J,K displayedweakerstainingintheareaofthepremusclemasses,indicatingthepresenceofunstainedcells.Figures (M),(N)and(O)showdorsalmuscleregionsstainedwithX-gal(M),MyoD(NO)andmyogenin()antibodies. Manycellswerenegativeforeachmarker.Inall,Lbx1/Lmx1bdoublelabelingexhibitednoLmx1b expressionin myogenicprecursorcells.Innewborns,itseemedpossiblethatLmx1b stainsonlyconnectivetissuecomponents butnodifferentiatedmusclecellsin()M .NB-newborn B9Figures 3.2.2.1Embryonicmammaryglanddevelopment 3.2.2.5K14,K18andLmx1bantibodystaining

A E11.5B E11.5C E13.5

D E15.5 E NewbornF Newborn

Figure2.6:MammaryglandprimordiaexpressLmx1b fromtheverybeginningand maintainexpressionthroughoutdevelopment. Byembryonicday11,thebeginningofmammaryglanddevelopment,firststainingwasdetected(A,B).Afterthe formationofalensshapedstructure,onlyepitheliallyarrangedcellswereLmx1b positive(C,D).Thispattern persisteduntilbirth(E,F).X-galstainingonsectionscounterstainedwithhematoxylinand/oreosin(A-F).

G H

K14/TOPROK18/TOPRO I J

Lmx1b/DAPILmx1b/DAPI

Figure2.7:Exclusively,luminalepithelialcellsexpressLmx1b inthemammarygland. Keratin14labeledspecificallymyoepithelialcells(confocalimagein(G)),whereaskeratin18wasemployedto markluminalepithelialcellsin(confocalimagein(H)).Comparisonofß-gal(=Lmx1b)antibodystaining(non- confocalimagesin(I,JH))tokeratin18()revealedthatbothmarkersexhibitanidenticalpattern. Lmx1b expressionwasrestrictedtoluminalepithelialcellsinthemammaryglandatallstages B10Figures 3.2.2Wholemountmammaryglands

A P26B P43

C P67D E9.5p

E E13.5pF E15.5p

G E18.5pH 1stLac

I 10thLacJ 4thWea

K 10thWea

Figure2.8:Lmx1b isexpressedthroughoutallstagesduringmammaryglanddevelopment. Examinationofmammaryglandsatdifferenttimepoints:postnataldevelopmentwithonlyafewordersof branchingatP26(A),pubertaldevelopmentatP43(BC)andP67()withexpansionoftheLmx1b positive parenchymatothemarginofthefatpad.Duringpregnancy(day9in(DEF),day13in(),day15in()andday18 in(G)),massiveproliferationincreasedtheepitheliatoadipocyteratio,andinthesamewayLmx1b expression wasregulated.Duringlactation(H,I),strongstainingwasmaintained,decreasingduringweaning(J,K)as involutionoccurred.Lmx1b wasexpressedatalltimepointsofdevelopmentdependingonthestatusofthe parenchyma,andnoup-ordownregulationwasobserved.AllmammaryglandswereclearedinBABB. B11Figures 3.2.2Mammarygland:Detailsofwholemounts

A P26B P67C P67

D E15.5pE E15.5pF E18.5p

G NBH 10thLacI 10thLac

J 4thWeaK 4thWeaL 10thWea

Figure2.9:Ateverystageofdevelopment,parenchymalcellswereLmx1b positive. Detailsofwholemountmammaryglandsatdifferentstagesofdevelopment.Secondary,tertiaryductsand terminalendbudswereformedatP26(AB)andsproutingofaductisshownin()atP67whentheparenchyma reachedthemarginofthefatpad(CD).Lymphnodesweredevoidofstaining().Increasingdensityofthe Lmx1b positiveparenchymawasobservedduringpregnancy(D-G),culminatingwiththeformationofsecretinglobules duringlactation(H,I).Involutionduringweaning(J-L)returnedthemammaryglandandLmx1b expressiontoa similarstateasbeforepregnancy. B12Figures 3.2.2.3MammaryglandsectionsI:P34topregnancy

A P34B P34C P34

D P34E E13.5pF E13.5p

G E13.5pH E13.5pI E15.5p

J E15.5pK E15.5p L E15.5p

K14/TOPRO

Figure2.10:Onlyluminalepithelialcellsexpress.Lmx1b SectionsofmammaryglandsatdifferenttimepointsrevealedthatLmx1b expressionwasrestrictedtoluminal epithelialcellsduringallstagesofdevelopment(P34in(C,D),atday13ofpregancyin(HI),day15in(). Allcellsinterminalendbudsexhibitedstaining(B).Luminalepithelialcellswereroundedandcuboidal(arrow in(DD))whilemyoepithelialcellswereelongated(arrowheadin()).Increasingdensityoftheparenchyma duringpregnancy(day13in(E-H),day15in(I-L))wasobserved,goingalongwithexpansionofX-galstaining withininthefatpad.In(KL)and(),alveolarunitswerecompared:X-galstainingmarkedluminalepithelialcells in(K).K14(green,Dapiinblue)labeledmyoepithelialcellsinconfocalimagein(L).Thetwoimagesappear like apositive(K)andnegative(L)slide.Imagesfrom(A)to(K)werestainedwithX-galandeosinand()D,E,H werecounterstainedwithhematoxylin. B13Figures 3.2.2.4MammaryglandsectionsII:Lactationtoweaning

A 10thLacB 10thLac

C 10thLacD 4thWea

E 4thWeaF 10thWea

G 10thWea

Figure2.11:Lmx1b expressionismaintainedduringlactationandweaning. Atthe10thdayoflactation(A-C),notallpartsofthemammaryglandwerestainedatthesamelevel(compare strongstainingin(A)andweakstainingin(CB)).Onhighpowermagnification,allcellswerepositive(). Differenceswereascribedtodifficultiesoffixationduetohighertissuedensity.Duringinvolutionmany parenchymalcellsundergoapoptosisdecreasingthepercentageofLmx1b positivecellsinthemammarygland (D-G).Theexpressionpatternshowingstainingonlyinluminalepithelialcellswasnotalteredduringlactation andweaning.AllsectionswerestainedwithX-galandeosin(exceptD). B14Figures 3.2.3Lmx1b expressioninthekidney

A B C

E14.5E17.5P26 D E F

E11.5E15.5E15.5 G H I

AdultAdultAdult

J TOPROK Lmx1bL WT-1/Dapi

M WT-1N Lmx1b/WT-1/TOPROO Lmx1b/Dapi

Figure2.12:Lmx1b isexpressedinalldevelopmentalstagesofthekidneyandrestrictedto WT-1positivepodocytesintheglomerulum. In(A-I),embryonicandadultkidneysfromLmx1b3'LacZ micewerestainedwithX-gal,counterstainedwith hematoxylin(G)andeosin(D-ID).FirststainingwasdetectedatE11.5inthemesonephros().Inthedeveloping kidney,expressionwasfoundatanystage(overviewwholemountsatE14.5in(AB),atE17.5in(),sectionsat E15.5in(E,F))andpersistedinadults(wholekidneyatP26in(C),sectionsofadultsin(G-I)),alwaysbeing restrictedtocellsoftheglomeruluminthecortexofthekidney.Inordertodeterminewhichkindofcellsexpress Lmx1b,antibodystainingwasperformedinaP25kidneywithß-gal(=Lmx1b)(K,O)andWT-1,apodocyte- specificmarker(M,LN).Allcellswerefoundtocoexpressbothgenes().Therefore,Lmx1b expressioninthe kidneyispresentatalltimesandrestrictedtopodocytes. B15Figures 3.2.4.2.1Lmx1b expressioninadultandembryonicorgansI

A B C

EosinEosinH&EEosin D E F

H&EEosinH&E HeartLiverSpleen

G H I

EosinEosinEosin J K L

EosinEosinH&E EmbryoniclungAdultlungAdultlung

Figure2.13:Lmx1b isexpressedintheembryonicandadultlungbutnotinheart,liverandspleen. SectionsofseveralorgansobtainedfromLmx1b3'LacZ micewerestainedwithX-galandcounterstainedwith eosinonlyorhematoxylinandeosin.Successofstainingwascontrolledbysectionsofthekidneyfromthesame animal,processedinthesameassay. Toppanel:Heart(A,D),liver(B,E)andspleen(C,F)werenegativeatallstagesexamined. Bottompanel:Intheembryoniclung(E15.5,G,JK)positivecellsweredetected.Inadultlungs(-overview), clustersof5to10cellsinarow(H,L)werepositive,alwaysbeing locatedintheprimarybronchiolesand terminalbronchioles. B16Figures 3.2.4.2.2Lmx1b expressioninadultandembryonicorgansII

A B

C D

UterusStomach

E F G

E10.5E10.5E11.5

Figure2.14:AdultstomachanduterusdisplayedthesamestaininginLmx1b3'LacZ miceand inwild-typecontrols.AtembryonicstagesLmx1b isexpressedintheforegut. Adultstomachs(B,D)anduteri(A,C)fromLmx1b3'LacZ weresectionedandstainedwithX-galand counterstainedwitheosin(A-G)andhematoxylin(C,D). Partofthewalloftheuterus(A,C)wasstained,butonlyindistinctregionsofthewholeorgan.Nostaining wasdetectedintheplacenta.Inthestomach,onlythebaseofgastricpitswasstained(B,D).Thesame patternwasfoundincontrolsof SwissWebsterwild-typeadultmice.

Atembryonicstages,foregutsshowedstainingclosetothelumenofductsatstageE10.5(E),high magnificationin(FG)andatstageE11.5in()whichhasalsobeendescribedinchick(Riddleetal.1995). B17Figures 3.2.4.3WholemountLmx1bexpressioninaP26LacZ/LacZ mouse

A B C

D E F

G H I

J K L

M N O

Figure2.15:AnalysisofwholemountX-galstainingofa26-dayoldLmx1b3'LacZ/LacZ mouse. Thefurandthebrainwereremoved,theperitoneumopenedandthewholemousewasstainedwithX-gal. Overview(A)oftheskullfromcranialshowedstaininginthemiddleear(BCandhighmagnificationin),inthe meninges(D),theeye(EF)andinterestinglyinthesuturesoftheskull().Lmx1bKO-/- showedsevereskull defects(Chenetal.1998).Theoverviewoftheabdomenin(G)revealednostaininginthespleenandliverbutthe kidneyswerepositive(G,H)andstainingwasalsodetectedinthefemalereproductivesystem:intheovary,inthe uterinetube(H,J)andintheuterus(I),intheureter(JK)andinretroperitoneallymphnodes().Inskeletal structures,themedialpartsoftheribs(L)andspinesofthevertebrawerestainedlikethesutures(FM).()showed partofasubmandibularglandandin(N),thefurisdisplayed. B18Figures 3.3.1MicromasscultureofLmx1b3’LacZ limbmesenchyme

A B C

2hours14hours24hours D E

Alcianblue 36hours50hours72hours Magnification:10x

F G H

2hours14hours24hours I J

36hours50hours Magnification:20x

Figure3.1:Dorsalandventrallimbmesenchymecellsremainmixedinamicromassculture. LimbmesenchymefromLmx1b3’LacZ micewasdispersedandculturedaccordingtothestandardsof micromassculturefor2,14,24,36and50hours.X-galstainingshowedthedistributionofdorsallimb mesenchymecells.Dorsalcellsdidnotsortoutfromventralcells.Alcianbluestainingexhibitedformationof cartilageafter72hoursofculture,indicatinggoodcultureconditionsandexplaininglossofcomplete homogenityafterlongerincubationtimes.Magnification:toppanel10x;bottompanel20x B19Figures 3.3.2Graftingandcultureoflimbmesenchymefromtransgenicmice 3.3.3EffectsofWnt7aandFGF8bapplicationonmouselimbs

A

B C

Figure3.2:VentralmesenchymegraftedunderneaththeAERkeepsitsnon-expressingstatus. BlocksofventralmesenchymefromLmx1b3'LacZ mice(E11.5)weregraftedunderneaththeapicalectodermal ridge(AER)ofwild-typeSwissWebstermice(E11.5),culturedfor26hoursandstainedwithX-gal(A).Grafted ventralcellsdidnotrevealanystaininginthedistalpartofthelimb(C)andthereforedidnotturnon Lmx1b expressionundertheinfluenceoffactorsinthedistallimb(6/6samples).Stainingwascontrolledbyattachinga pieceofLmx1b-positivedorsalmesenchymeonthecontralateralnon-manipulatedlimb(B). Magnification:A:3X;B,C:7X

D E F

G H I

Figure3.3:ProximalventrallimbmesenchymedidnotrespondtoWnt7aandFGF-8bapplication. EctodermwasremovedfromLmx1b3'LacZ mouselimbsatE11.0.Ventrallimbmesenchymereceivedeither beadssoakedinFGF-8borwascoveredwith3T3-Wnt7acellsorbothproteinswereapplied(D,E,G,H).Limbs exhibitedagoodmorphologyafter26hoursofculture.TissuewasfixedandstainedwithX-gal.Nochangeof expressionwasobserved(14/14)intheventralmesenchyme:dorsalviewin(DandE),ventralviewin(GHand). Insomecases(5/15),distalmesenchymeeitherlostLmx1b expressionand/orgrowingcellsdidnotturnonthe gene(FI).3T3-Wnt7asecretingcellsdidnotshowanyX-galstaininginacontrolculture(). B20Figures 3.3.4Inductionofventralproximallimbmesenchyme

A B C

D E F

Figure3.4:VentralwingmesenchymedoesnotturnonLmx1b whengraftedtovariousdorsal positions. Ventralproximalmesenchymeblocks(OPA)fromaquaildonoratstageHH22-24wereinsertedinto differentlocationsonthedorsalpartofachickhostwingatHH20-23,reincubatedbetween8and22hours andanalyzedbywholemountinsituhybridizationfor.Lmx1b Inallsamples(16/16),dorsalgraftswerenotstainedbyinsituhybridizationinwholemountembryos(A,B, D,E)andonsections(CF).()showsacontralateralcontrollimb.

G H

Figure3.5:Insituhybridizationanalysisconfirmedthatventralmesenchymemaintainsitsnon- expressingstatuswhengraftedtodorsal. VentralmesenchymeblocksweregraftedtodorsalatHH22(seeoperationschemeOPA),incubatedfor26hours andprocessedforsectioninsituhybridizationforLmx1b(blue). GraftedquailcellsweredetectedbyQCPN antibodystaining(Cy3-red)andimagesoverlayeddigitally.Thegraftwascompletelysurroundedby-Lmx1b positivedorsalmesenchyme(G).Cellsmigratedwithinthedorsalpartofthelimbmainlytowardsdistalbutalso toproximalpositions(H).Allgraftedquailcellswithinthegraftandallmigratingcellsmaintainedtheirnon- expressingstatus(G,H). B21Figures 3.4.1Lbx1 expressioninthequailandchickwing

A B C

Quail-Lbx1QuailcontrolMouse-Lbx1

D’ D E

Lbx1Lbx1Pax3

F G H

Lmx1Lbx1Lbx1 I J

Lbx1Lbx1

Figure4.1:Inquailandchickwings,moreLbx1-positivemyogenicprecursorcellsarelocated intheventralpremusclemassthaninthedorsalone. Figures(A,CB)werestainedwithLbx1antibodies,in()theprimaryantibodywasomittedascontrol. Counterstainingin(A,B)withDapi. Inquailandchickwings(A),moreLbx1 positivecellswerefoundintheventralpremusclemassthanintheinthe dorsalone.Incontrast,Lbx1 stainingonE11.5mouselimbs(C)showedtobethesameintheventralanddorsal premusclemass.Theprimaryantibodywasomittedinacontrol(B)inwhichonlysomefluorescenterythrocytes weredetectedinthecentralpartofthewing. (D-I)insituhybridizationusingcLbx1(D,G-J),Lmx1(FE)andcPax3 ()antisenseprobesonchickembryosat HHstages19(GE)toHH25().ComparisonofLbx1andPax3 expressionexhibitedastrongerventraldomainin (D’andD)implyingthatmoremyogenicprecursorsarepositiveforLbx1(DE)thanforPax3 ()intheventral premusclemassofthechickwing.(G-I)wholemountinsituhybridizationshowedLbx1 stainingfromdifferent directions.Thehindlimbin(JI),ahighmagnificationof(),displaysadorsalviewofLbx1 limbexpression. ComparisonofLmx1(F)(notethestronglypositivemesonephros)toLbx1(G,HE)andPax3 ()expression patternsshowedaventralpremusclemass(pmm)locatedentirelyintheLmx1-negativemesenchymeanda dorsalpmmlocatedcompletelywithinthedorsalLmx1-positivepart. B22Figures 3.4.2Interspecificgraftingexperiments-tableI

A OPAB OPA

C OPBD OPB

E OPCF OPD

G OPDH OPD

* * *

Figure4.2: Migrationbehaviorofgraftedcellsdependedonthedonor-hostagerelationshipandthe position. InasectionanalysisofquailtochickgraftingexperimentsquailcellsweredetectedwithaQCPNantibody(red) inchickwingsandcounterstainedwithDapi(blue). Graft(AB)andmigratingcells()inthedorsalpartofthewingafteroperationA(ventraltodorsal).Mostcells migratedintotheareaofthefuturepectoralmusclefromgrafts(C)insertedintotheproximalventralregion (operationB)asshownin(DD).Fewercellsmigrateddistallywithintheventralpremusclemass(arrows). Ifthedonor-hostagerelationshipwasnottakenintoconsiderationorgraftsweretakenfromveryproximal positions,nomigrationwasfound:homotopicventralproximaloperationCshowninpanel(E).OperationD dorsalhomotopicshowedthatindorsalgrafts(G,H),mostcellsmigratedproximally,presumablysupplying myogenicmaterialfortherotatorcuff.(F)Nervefibers(arrow)dividedagraftafteroperationD. *=erythrocytes,autofluorescence B23Figures 3.4.3Interspecificgraftingexperiments-tableII

A OPEB OPE

*

C OPED OPE

E OPFF OPD *

* *

G OPEH OPA

*

Figure4.3: Migratingcellsstayeitherdorsalorventralandtakedistalandproximaldirections. SectionanalysisofquailtochickgraftingexperimentsdetectingquailcellswithaQCPNantibody(red)inchick wingscounterstainedwithDapi(blue).Chickwingshavingreceivedagraftintheprogresszonefromventral proximalinoperationEexhibitedstrongmigrationinthedorsalandventralpremusclemass(A,C,D). Highmagnificationconfocalimageofaventralgraft(B)showingcellsmigratingtoproximalanddistal positions.Cellsfromboth,dorsalandventralpremusclemassessuppliedmyogenicmaterialford-v-segment overlappingmuscles(lineinDE)(n=1).Intwosampleshavingreceivedacentral(OPF-)andadorsalgraft(OP D-F),migratingcellswerefoundinaveryproximalpositionofthewinginacentralpartindicatingthatthere couldbeexchangeinthisregion(„lbxnose“)toformamuscleofthemusculotendinouscuff. Graftedcellsintegratedinachondrogeniczonedidnotmigrate(G)unlessthegrafthadpartialaccessto mesenchymeallowingcellularmovement.Fewcellsmigratedtowardsproximalpositionsfromasmallgraft inserteddorsally(operationA-H).Magnification:20xinpanelD,10xinpanelA,C,E,F,GandH; *=erythrocytes,autofluorescence B24Figures 3.4.2Interspecificgraftingexperiments-tableIII

A OPAB OPA

C OPCD OPC

* * E OPCOPEF OPE G

H OPFI OPF

Figure4.4:Migratingcellsstayeitherdorsalorventralandtakedistalaswellasproximal directions. QCPNantibodystaining(red)onchickwingsectionsofHH27embryosinthedorsoventralplain.Ventralquail mesenchymeblocksweregraftedintodifferentlocationsinchickhosts. Cellsgraftedintothedorsalpartofthelimb(operationA)showedstrongmigrationtodistal(A)andless migrationtoproximal(B)positions.(C,D) showdifferentplainsofsectionfromthesamewingafterahomotopic ventralgraftingprocedure.Manycellsmigrateddistallywithintheventralpremusclemass(D,positionofthe graft=asterisk)andalsostronglytoproximalpositions(C -asterisk).Highmagnificationofquailcellswithin ventralpremusclemass(EG).Insertionintotheprogresszone()madecellsmigrateinadorsalandventral pathwaybutnocellsweredetectedincentralregions(arrowinF).Centralgraftscompletelysurroundedby formingcartilagedidnotshowanymigration(H,I).Allembryoswerereincubatedfor48hours.