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«In a u g u r a l-D isse r t a t io n zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln ...»

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Isoform-specific knockout of the Neuregulin-1 gene

In a u g u r a l-D isse r t a t io n


Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Universität zu Köln

vorgelegt von

Li Li

aus Guizhou, China

Berichterstatter: Prof. Dr. Walter Doerfler

Prof. Dr. Carmen Birchmeier

Tag der Disputation: 29.11.2002



1.1. Neuregulin-1 1

1.2. Receptors of Neuregulin-1 2

1.3. Isoforms of Neuregulin-1 4

1.4. Functions of Neuregulin-1 in the heart 5

1.5. Functions of Neuregulin-1 in the neural crest and glial cell lineages 6

1.6. Sox10 and its functions in the development of glial cells 8

1.7. The olfactory system and the development of the primary olfactory path 9

1.8. Odorant receptors and olfactory signal recognition 11

1.9. The olfactory topographic map and olfactory axon guidance 11

1.10. Olfactory glial cells 12

1.11. The purposes of the project 13


2.1. Abbreviations 14

2.2. Materials 15 2.2.1. Bacterial strains 15 2.2.2. Vectors/plasmids 16 2.2.3. ES cell line 16 2.2.4. Primary antibodies 16 2.2.5. cDNA probes for in situ hybridization 17 2.2.6. Mouse strains 17

2.3. Methods: Generation of the knockout mouse strain 18 2.3.1. Molecular biological techniques 18 2.3.2. Site specific mutagenesis with PCR 18 2.3.3. ES cell culture, electroporation and neomycin-resistant selection 19 2.3.4. Identification of homologous recombinant clones 20 2.3.5. Cre transient expression 20 2.3.6. Microinjection of blastocysts

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1. Introduction

1.1. Neuregulin-1 Neuregulins are a family of Epidermal Growth Factor (EGF)-like ligands that signal via receptor tyrosine kinases of the ErbB family. First identified as an activity able to promote the proliferation of glial cells (Glial Growth Factor, GGF; Raff et al., 1978; Brockes et al., 1980;

Lemke and Brockes, 1984), Neuregulin was independently characterized as a factor that induces expression of acetylcholine receptors in cultured myotubes (Acetylcholine-Receptor-Inducing Activity, ARIA) and promotes the differentiation of developing muscle cells in vitro (Jessell et al., 1979; Falls et al., 1990). Later, in other independent studies, the factor was characterized as an activity that stimulates the phosphorylation of the ErbB receptor neu/HER2/ErbB2, and named Neu Differentiation Factor (NDF; Peles et al., 1992) or Heregulin (HRG; Holmes et al., 1992). The cloning of the cDNAs encoding NDF (Wen et al., 1992), HRG (Holmes et al., 1992), ARIA (Falls et al., 1993) and GGF (Marchionni et al., 1993) finally revealed that these proteins are encoded by a single gene. The presence of multiple promoters and extensive alternative splicing of mRNAs account for the diversity of isoforms encoded. The consensus name used today for the various isoforms is Neuregulin-1 (NRG1) (Marchionni et al., 1993).

In vertebrates, three additional Neuregulin genes, Neuregulin-2 (Busfield et al., 1997; Carraway et al., 1997), Neuregulin-3 (Chang et al., 1997; Zhang et al., 1997) and Neuregulin-4 (Harari et al., 1999) have been identified (Fig. 1a). The encoded proteins share high sequence homology with Neuregulin-1 in their EGF-like domains (Fig. 1b). The EGF-like domain that contains about 50-60 amino acids is characterized by three pairs of cysteines, which are important for tertiary structure and biological function. In Neuregulin-1s, the EGF-like domain is essential to elicit receptor binding, and can on its own cause receptor heteromerization, tyrosine phosphorylation and downstream signal activation (Holmes et al., 1992). In addition to EGF and the Neuregulins, the family of EGF-like factors also includes Transforming Growth Factor-α (Kumar et al., 1995), Amphiregulin (Shoyab et al., 1989), Heparin-binding EGF-like Growth Factor (Higashiyama et al., 1991), Betacellulin (Shing et al., 1993) and Epiregulin (Toyoda et al.,

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Figure 1. Schematic structures of the four Neuregulin ligands and alignment of their EGF-like domains (a) All Neuregulins (NRG1-4) possess an EGF-like domain (red, EGF) that is essential for receptor binding and function.

α- and β-variants of the EGF domain have been characterized for NRG1 and NRG2. Three major isoforms (types I-III) of NRG1 are produced by the usage of different promoters and alternative splicing. Other domains present in Neuregulins include Ig-like domains (Ig), domains rich in potential glycosylation sites (glyco), transmembrane domains (TM, black box) and cytoplasmic domains (wavy lines). Kringle-like (kringle) and cysteine-rich domains (CRD) are found in types II and III NRG1, respectively. Signal peptides or internal hydrophobic sequences are indicated as gray shaded boxes (Hy). (b) All Neuregulins share high sequence homology in their EGF-like domains, which are characterized by three pairs of cysteines (green). Amino acids identical or which are conservatively substituted in all Neuregulins are marked in gray. The α- and β-spliced variants of the EGF-like domain that have been characterized for NRG1 and NRG2 are boxed. (Adapted from Garratt et al., 2000; Buonanno and Fischbach, 2001).

1.2. Receptors of Neuregulin-1 Neuregulins signal through ErbBs, a family of receptor protein tyrosine kinases related to the receptor for Epidermal Growth Factor (EGFR). Four ErbB receptors, ErbB1/HER1/EGFR, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4 (170-185 kDa), are encoded by the mammalian genome. They all contain two cysteine-rich domains in their extracellular parts, a single transmembrane domain and a relatively large cytoplasmic portion containing 8-18 tyrosine residues at the C-terminal tails (Fig. 2). While the extracellular parts of the receptors function in ligand binding, their cytoplasmic portions are responsible for their tyrosine kinase

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heteromerization and the phosphorylation of tyrosine residues in their cytoplasmic portions. The phosphorylated tyrosine residues serve as docking sites for various adapter proteins and other signaling molecules, which constitute a molecular signaling cascade and mediate further propagation of the signal.

Figure 2. Schematic structures of the Neuregulin-1 receptors ErbB2, ErbB3 and ErbB4 The ErbB receptors contain two cysteine-rich domains, located extracellularly (ovals), a transmembrane domain and a cytoplasmically located tyrosine kinase domain (oblongs).

Ligand binding stimulates heteromerization of the receptors and phosphorylation of tyrosine residues, located principally in the tails of the molecules. Whereas ErbB2 (blue) and ErbB4 (brown) have catalytically active tyrosine kinase domains, the corresponding domain in ErbB3 (green) possesses no or very little activity (hatched). ErbB2 acts as a co-receptor in transduction of Neuregulin-1 signals, in neural crest tissues through heteromerization with ErbB3, and in the heart, with ErbB4.

(Adapted from a drawing of Stefan Britsch, C. Birchmeier laboratory).

Neuregulin-1 binds the ErbB3 and ErbB4 receptors directly. In contrast to the other ErbBs, ErbB3 exhibits no catalytic activity, as it lacks specific amino acid residues that are conserved in all other protein tyrosine kinases (Guy et al., 1994). ErbB2, on the other hand, is a receptor without any known direct ligand (Klapper et al., 1999), and is activated by Neuregulin-1 through heteromerization with ErbB3 or ErbB4 (Carraway and Cantley, 1994; Plowman et al., 1993;

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Neuregulin-1 receptor heteromers are essential for Neuregulin-1 signaling, the ErbB2/ErbB3 and the ErbB2/ErbB4 heteromers. The presence of ErbB2 in the receptors changes receptor affinities and signaling properties. ErbB2 stabilizes ligand-bound ErbB3 and ErbB4 receptors by reducing the rate of ligand dissociation, as well as increasing by 100-fold the ligand binding affinity (Fitzpatrick et al., 1998; Jones et al., 1999). ErbB2 is also a strong activator of the MAP kinase pathway. Thus, ErbB2-containing heteromers are stable, and have potent signaling properties (Graus-Porta et al., 1995; Graus-Porta et al., 1997; Karunagaran et al., 1996; Tzahar et al., 1996; Pinkas-Kramarski et al., 1996; Riese et al., 1995; Riese et al., 1996a; Riese et al., 1996b; Kokai et al., 1989).

1.3. Isoforms of Neuregulin-1 The Neuregulin-1 gene has been extensively characterized. Usage of different promoters and alternative RNA splicing of exons give rise to at least 15 different isoforms (reviewed by Lemke, 1996). The various isoforms of Neuregulin-1 were originally named based on the distinct biological assays used for their identification. These isoforms include NDF, HRG, GGF, ARIA (mentioned above) and also SMDF (Sensory and Motoneuron-Derived Factor), which was identified later (Ho et al., 1995; Yang et al., 1998). The isoforms can be classified into three major types that are produced from three distinct promoters and contain distinct N-terminal sequences (Fig. 1a) (Meyer et al., 1997; Fischbach and Rosen, 1997). Type I Neuregulin-1 (NDF, HRG, and ARIA) contains an immunoglobulin (Ig)-like domain, a sequence stretch that contains many glycosylation sites, a transmembrane domain and a cytoplasmic portion. Type II Neuregulin-1 (GGF) contains a signal peptide, a GGF-specific (kringle-like) domain and an Ig-like domain. Type III Neuregulin-1 (SMDF) contains a cysteine-rich domain or SMDF domain, as well as the transmembrane domain and C-terminal part present in type I Neuregulin-1. Alternative splicing at the C-terminal region of the EGF-like domain gives rise to α- and β-variant isoforms of Neuregulin-1 (Holmes et al., 1992). Type I Neuregulin-1 contains either the α- or β-variant of the EGF-like domain, denoted as type Iα and type Iβ isoforms.

Types II and III Neuregulin-1 contain only the β-variant of the EGF-like domain (Meyer et al.,

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type Iβ are produced, with the exception of the mammary gland that produces only type Iα Neuregulin-1 (Yang et al., 1995).

All isoforms of Neuregulin-1 bind directly to both ErbB3 and ErbB4 receptors and can induce the formation of ErbB2/ErbB3 or ErbB2/ErbB4 receptor heteromers (Crovello et al., 1998;

Pinkas-Kramarski et al., 1998; Tzahar et al., 1997; Harris et al., 1998). However, the α- and β-isoforms distinguished by their C-termini of the EGF-like domains differ each other dramatically in their receptor binding affinities. The isoforms containing the β-variant EGF-like domain have a much higher binding affinity for ErbB3 or ErbB4 homodimers than α-variant isoforms in a cell free system (Jones et al., 1999). The β-isoforms are also superior to the α-isoforms in eliciting responses in cellular systems that express different combinations of ErbB receptors (Pinkas-Kramarski et al., 1998). Although signaling by both α- and β-isoforms is funneled through MAP kinase, their distinct affinities for the receptors cause differential activation of the MAP kinase pathway, resulting in discrete biological effects (Pinkas-Kramarski et al., 1998). β-isoforms have significantly higher potency and elicit longer lasting cellular responses than α-isoforms.

In vivo, the different major isoforms of Neuregulin-1 have distinct spatial and temporal expression patterns and take over different biological functions (Meyer et al., 1997). During development, type I Neuregulin-1 is the earliest detectable isoform and is expressed in the endocardium. Type II Neuregulin-1 is expressed late and produced mainly in the nervous system. Type III Neuregulin-1 is the major Neuregulin-1 isoform produced by sensory and motoneurons, and is also expressed in the brain (Wen et al., 1994; Meyer and Birchmeier, 1994).

1.4. Functions of Neuregulin-1 in the heart Mouse mutants homozygous for null mutations in NRG1 (Meyer and Birchmeier, 1995), ErbB2 (Lee et al., 1995; Erickson et al., 1997; Britsch et al., 1998) and ErbB4 (Gassmann et al., 1995)

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