“I love fools´ experiments. I am always making them.” -- Charles Darwin

Faculty opponent: William J. Pavan, Professor,

National Institutes of Health, Bethesda, MD, USA

Examining committee: Per Jemth, Associate Professor,

Uppsala University, Uppsala, Sweden Lena Åslund, Senior lecturer, Uppsala University, Uppsala, Sweden Anna Qvarnström, Researcher, Uppsala University, Uppsala, Sweden

Chairman: Erik Fries, Professor, Uppsala University, Uppsala, Sweden Supervisors: Leif Andersson, Professor, Uppsala University, Uppsala, Sweden Susanne Kerje, Researcher,

Uppsala University, Uppsala, Sweden

Klas Kullander, Professor, Uppsala University, Uppsala, Sweden

List of Papers

This thesis is based on the following Papers, which are referred to in the text by their Roman numerals.

I Gunnarsson, U.*, Hellström, A.R.*, Tixier-Boichard, M., Min-

vielle, F., Bed’hom, B., Ito, S., Jensen, P., Rattink, A., Verei-jken, A., Andersson, L. (2007) Mutations in SLC45A2 Cause Plumage Color Variation in Chicken and Japanese Quail. Ge-netics 175:867-77.

II Hellström, A.R., Sundström, E., Gunnarsson, U., Bed’hom, B., Tixier-Boichard, T., Honaker, C.F., Sahlqvist, A-S., Jensen, P., Kämpe, O., Siegel, P.B., Kerje, S., Andersson, L. (2010) Sex-linked barring in chickens is controlled by the CDKN2A/B tu-mour suppressor locus. Pigment Cell Melanoma Res. 23:521-30.

III Hellström, A.R., Watt, B., Shirazi Fard, S., Tenza, D., Kerje, S., Mannström, P., Ulfendahl, M., Hallböök, F., Kullander, K., Raposo, G., Marks, M.S., Andersson, L. (2010) Inactivation of the Silver gene alters shape of eumelanosomes but has only a subtle effect on pigmentation. Manuscript.

* These authors contributed equally to the work.

Reprints were made with permission from the respective publishers.

Related work by the author

Wahlberg, P., Strömstedt, L., Tordoir, X., Foglio, M., Heath, S., Lechner, D., Hellström, A.R., Tixier-Boichard, M., Lathrop, M., Gut, I.G., Andersson, L. (2007) A high-resolution linkage map for the Z chromosome in chicken re-veals hot spots for recombination. Cytogenet Genome Res. 117:22-9.

Hansson, C.M., Buckley, P.G., Grigelioniene, G., Piotrowski, A., Hellström, A.R., Mantripragada, K., Jarbo, C,. Mathiesen, T., Dumanski, J.P. (2007) Comprehensive genetic and epigenetic analysis of sporadic meningioma for macro-mutations on 22q and micro-mutations within the NF2 locus. BMC Genomics 12;8:16

Lagerström, M.C., Rabe, N., Haitina, T., Kalnina, I., Hellström, A.R., Klovins, J., Kullander, K., Schiöth, H.B. (2007) The evolutionary history and tissue mapping of GPR123: specific CNS expression pattern predomi-nantly in thalamic nucleic and regions containing large pyramidal cells. J Neurochem. 100:1129-42

Lagerström, M.C., Hellström, A.R., Gloriam, D.E., Larsson, T.P., Schiöth, H.B., Fredriksson, R. (2006) The G protein-coupled receptor subset of the chicken genome. PLoS Comput Biol. 2:e54.

Contents

Introduction................................................................................................... 11  Pigmentation ............................................................................................ 12  

Melanocyte development .................................................................... 12  Two types of melanosomes ................................................................. 14  Disease related pigmentation............................................................... 15  

Model organisms...................................................................................... 16  Chicken (Gallus gallus)....................................................................... 16  Japanese quail (Coturnix japonica) ..................................................... 17  Mouse (Mus musculus)........................................................................ 18  

Forward genetics ...................................................................................... 18  Genetic variation ................................................................................. 18  Pedigrees ............................................................................................. 19  Linkage analysis .................................................................................. 20  IBD mapping and mutation detection ................................................. 21  

Reverse genetics....................................................................................... 23  Generation of mouse knockouts .......................................................... 23  

Aims of the thesis ......................................................................................... 25  

Present investigation..................................................................................... 26  Forward genetics – mapping of monogenic traits (Papers I and II)......... 26  

Background ......................................................................................... 26  Results and discussion......................................................................... 29  

Reverse genetics – generation and characterization of a PMEL17 knockout mouse (Paper III)...................................................................... 32  

Background ......................................................................................... 32  Results and discussion......................................................................... 33  

General discussion and further perspectives................................................. 38  

Svensk sammanfattning ................................................................................ 40  

Acknowledgements....................................................................................... 42  

References..................................................................................................... 44  

Abbreviations

5’RACE Rapid amplification of 5’ complementary DNA ends ARF Alternative reading frame protein ASIP Agouti-signaling protein BCDO2 Beta-carotene dioxygenase 2 Bp Base pair CDKN2A/B Cyclin-dependent kinase inhibitor 2A/B cM Centimorgan CNV Copy number variation DCT Dopachrome tautomerase DNA Deoxyribonucleic acid EMSA Electrophoretic mobility shift assay ER Endoplasmic reticulum ES Embryonic stem HPRT Hypoxanthine-guanine phosphoribosyltransferase HSV-tk Herpes simplex virus thymidine kinase IHC Immunohistochemistry Kg Kilogram LD Linkage disequilibrium LOD Logarithm of odds MC1R Melanocortin receptor 1 MDM2 Mouse double minute 2 homolog MITF Microphthalmia-associated transcription factor mRNA Messenger RNA NC Neural crest NeoR Neomycin resistance gene OS Obese strain PAX3 Paired-homeodomain transcription factor 3 PMEL17 Pre-melanosomal protein 17 RJF Red junglefowl RNA Ribonucleic acid ROS Reactive oxygen species RPE Retinal pigment epithelium SCP Schwann cell precursor SLC45A2 Solute carrier family 45 member 2 SOX10 SRY-box containing transcription factor TF Transcription factor

TGN Trans-Golgi network TYR Tyrosinase TYRP1 Tyrosinase-related protein 1 TYRP2 Tyrosinase-related protein 2 WL White leghorn WNT1 Wingless/INT-related 1

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Introduction

The modern science of genetics was formed in the beginning of the 20th cen-tury, when the work of Charles Darwin (1809-1882) and Gregor Mendel (1822-1884) were widely accepted (Darwin, 1859; Mendel, 1866). Darwin (1859) published “On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life” in which he proposed his ideas that all species descend from common ancestors and that evolution is driven by natural selection. Seven years later, Mendel published his results from studies of simple traits in pea plants. From that work he could deduce the basic principles of inheritance, today known as the Men-delian laws of inheritance. The Deoxyribonucleic Acid (DNA) harbors the genetic code essential for all living organisms and the nitrogenous bases adenine (A), cytosine (C), guanine (G), and thymine (T) are its building blocks. In 1953, Watson and Crick discovered that DNA forms a helix, where the nucleotides form a polymer on a backbone of linked sugar and phosphate groups (Watson and Crick, 1953). The helix consists of two polymers that are anti-parallel, where A forms hydrogen bonds to T and C forms hydrogen bonds to G.

A more recent milestone was reached in year 2000, when the sequence of the human genome was announced at a press conference in the White House by US president Bill Clinton and the British Prime Minister Tony Blair. It took a large international scientific consortium, in parallel with the Celera Corporation, ten years to finish a draft sequence of the human genome and the price tag was around three billion US dollars (NHGRI, 2009). Since then, sequencing techniques have been improved and the costs involved have de-creased rapidly. Today, a large number of genomes from different species, individual human genomes, and even cancer genomes have been published and it is possible for a relatively small laboratory to carry out a genome-resequencing project both technically and financially.

In this thesis, genes of relevance for pigment cell biology were investi-gated. Geneticists have long studied coat color and patterning phenotypes as a model for Mendelian genetics. By following a monogenic mode of inheri-tance it has been relatively easy to identify the underlying gene and muta-tion, making color and patterning phenotypes valuable models for under-standing the function of these genes and their interaction with other genes. The biology and development of the pigment cell is well conserved between species and regulated by the same genes and pathways (reviewed in “Pig-

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mentation” of this thesis). Therefore, knowledge gained using chicken and mouse models is not only valuable for understanding the normal pigmenta-tion process in human, but also for understanding what goes wrong in dis-eases involving the pigment cell, e.g. skin cancers.

Pigmentation Melanocyte development In vertebrates all pigment cells originate and differentiate from the neural crest (NC), except for the retinal pigment epithelium (RPE) of the eye which derivates from the optic cup in the developing forebrain (Silver et al., 2006). NC stem cells are unique to vertebrates and arise from the dorsal neural tube in early development. These cells migrate along defined pathways to differ-ent sites of the embryo where they differentiate into distinctive cell types such as pigment cells, neurons, endocrine cells, smooth muscle, bone and cartilage (Le Douarin and Kalcheim, 1999).

The development of pigment-producing melanocytes is summarized in Figure 1 (black dots). The process starts with the melanoblasts precursors which are present in the NC at embryonic day 8.5 (E8.5) in mouse (Serbedzija et al., 1990). At this stage the cells express SRY-box containing transcription factor 10 (Sox10), Wingless/INT-related 1 (Wnt1), and Paired-homeodomain transcription factor 3 (Pax3), which are all key players in melanocyte development (Brewer et al., 2004; Goulding et al., 1991; Mol-laaghababa and Pavan, 2003). At E10.0-E10.5, the cells have developed into mature melanoblasts (the precursor to melanocytes) expressing the receptor tyrosinase kinase Kit and microphthalmia-associated transcription factor (Mitf) (Opdecamp et al., 1997; Wilson et al., 2004). Shortly thereafter, ex-pression of the melanogenic enzyme marker Dopachrome tautomerase (Dct) begins and the cells line up along the dorsal length of the neural tube, ready to begin their migration (Figure 1) (Pavan and Tilghman, 1994). The cells divide and travel along a dorsal-lateral pathway between the somites and the ectoderm and subsequently invade the dermis. This is opposite to the other NC stem cells, which migrate ventrally between the neural tube and the somite (Figure 1; gray dots). At E13.5, the melanoblasts colonize the epi-dermis (Le Douarin and Kalcheim, 1999). Finally, at E15.5 a number of melanoblasts migrate into the hair follicles and genes necessary for synthesis of pigment are expressed, such as Tyrosinase (Tyr) and Tyrosinase-related protein 1 (Tyrp1)(Figure 1). From this stage the melanoblasts have devel-oped into pigment synthesizing melanocytes (Silver et al., 2006). Moreover, precursor stem cells migrate to the bulged region of developing hair follicles and form a self-renewing pool from which new melanocytes can be recruited (Nishimura et al., 2002).

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Until recently, the above-described dorsal-lateral pathway was the general opinion of how NC derived melanocytes develop and migrate. In 2009, Schwann cell precursors (SCPs) of the growing nerve were reported to rep-resent a stem cell niche from which large numbers of skin melanocytes originate (Adameyko et al., 2009). The SCPs arise in the NC ventral migra-tory pathway and this finding may lead to the identification of new factors important for melanocyte development.

Figure 1. The development of pigment-producing melanocytes initiates from neural crest (NC) stem cells. These cells express Sox10, Pax3 and Wnt1 at E8.5. At E10.0-E10.5 the cells have developed into melanoblasts expressing Kit and Mitf. Shortly after Dct expression kicks in and cells line up along the dorsal length of the neural tube and begin to migrate. The cells divide and travel along the dorsal-lateral path-way between the somites and the ectoderm and subsequently invade the dermis in a manner opposite to other NC stem cells, which migrate ventrally between the neural tube and the somite. At E15.5 a number of melanoblasts migrate into the hair folli-cles and genes necessary for synthesis of pigment are expressed, such as Tyr and Tyrp1.

Melanin and/or melanocytes are also present in iris and choroid of the eye (Spritz, 1994), in the stria vascularis of the cochlea (Tachibana, 1999), in the leptomeninges (Goldgeier et al., 1984), substantia nigra and locus coerulus of the brain (Zecca et al., 2002), in the heart (Brito and Kos, 2008; Yajima and Larue, 2008), and in the adipose tissue of the lung (Randhawa et al., 2009). The pigment has several functions, from the display of camouflaging coloration and patterning of whole organisms, to protecting the skin and eyes from harmful UV-radiation. The function of melanin in the inner ear is under debate. On one hand, studies have shown that melanocytes and melanin are important for hearing and balance (Barrenas and Lindgren, 1990; Tachibana, 1999; Takeda et al., 2007; Tassabehji et al., 1994). On the other hand, stud-ies in albino mice have shown that whilst melanocytes are essential for hear-ing, they are not required to produce pigment. The neuoromelanin pigment

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in the brain accumulates with age and loss of pigment is linked with Parkin-son’s disease (Zecca et al., 2002). Melanocytes were recently shown to be present in the heart and in the lung of morbidly obese humans, however the cellular function is unclear (Randhawa et al., 2009; Yajima and Larue, 2008).

Two types of melanosomes Pigmentation is the result of the assembly of two types of melanin, brown/black eumelanin and yellow/red pheomelanin. Melanin is synthesized and stored in internal vesicle-like organelles, termed melanosomes, which are located in melanocytes. The melanosomes are believed to bud off from the endoplasmic reticulum (ER) (Akutsu and Jimbow, 1988). Eumelanin is synthesized in rod shaped eumelanosomes, whilst pheomelanin is synthe-sized in round pheomelanosomes (Figure 2). In the stage II eumelanosome, the cleaved product of Pre-melanosomal protein 17 (PMEL17), encoded by the Silver gene, forms a fibril matrix onto which the eumelanin is deposited in stage III-IV (Hearing, 2005; Kushimoto et al., 2003). The pheomelanin deposition in the pheomelanosome is weaker and is less organized due to the absence of a scaffold like the PMEL17 fibrils (Berson et al., 2003). Eumela-nosome development is dependent on high levels of Tyrosinase (TYR) ex-pression and moderate expression of the Tyrosinase-related-proteins 1 (TYRP1) and 2 (TYRP2), whilst pheomelanosome development requires low expression of TYR but the addition of cysteine (Kobayashi et al., 1995; Wakamatsu and Ito, 2002). TYR, TYRP1, and TYRP2 are believed to be transported from the trans-Golgi network (TGN) to stage II melanosomes by Solute carrier family 45 member 2 protein (SLC45A2) via vesicles (Figure 2). Once melanosomes are fully developed and pigmented they are trans-ferred to surrounding keratinocytes in the skin or to the hair shafts (or to the feather in birds). This process is not fully understood and various mecha-nisms including exocytosis, cytophagocytotis, fusion and membrane vesicle transport have been proposed (Van Den Bossche et al., 2006).

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Figure 2. Schematic picture illustrating the production of pheomelanosomes and eumelanosomes. Both organelles develop within the melanocyte and are transferred to surrounding keratinocytes, hair shafts or feather follicles. Premelanosomes bud off from ER. The proteins required for melanosome differentiation are transported via vesicles from the TGN. High levels of TYR, TYRP1 and TYRP2 induce eumelanosome progression, whilst low levels of TYR and the presence of cysteine lead to pheomelanosome progression.

Disease related pigmentation Mutations in genes involved in melanocyte development may lead to dis-ease. The uncontrolled growth of melanocytes results in melanoma, the least common but most deadly form of the three skin cancer types; melanoma, basal cell cancer, and squamous cell cancer. In USA, melanoma is the fifth and seventh most common cancer form in men and women respectively (Jemal et al., 2010). Metastatic melanomas are not responsive to chemo- or radiotherapy, therefore lesions must be removed at an early stage by surgery. The survival prognosis for patients with metastatic melanomas is poor; six to nine months (Ibrahim and Haluska, 2009).

Other diseases affecting the pigmentary system can be divided into four classes (Tomita and Suzuki, 2004), (i) disorders of melanoblast migration from the neural crest, usually leading to white patches of the skin, e.g.

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Waardenburg syndrome (MIM 193500) and Piebaldism (MIM 17280); (ii) disorders of melanosome formation and transfer to keratinocytes, e.g. Her-mansky-Pudlack syndrome (MIM 203300) and Chediak-Higashi syndrome (MIM 214500); (iii) disorders of melanin synthesis in the melanosome, e.g. Oculocutaneos albinism types 1-4 (MIM 203100, 203200, 203290, 606574); (iv) disorder of mature melanosome transfer in the melanocyte, e.g Griscelli syndrome (MIM 214450, 607624, 609227).

Model organisms The use of animal models in science to increase our understanding of basic biology is not new, Aristotle recorded his practice of experimentation on living animals in the fourth century BC (Fox, 1984). By dissecting the genet-ics underlying the immense phenotypic variation seen in nature, or those in laboratory mice, we can learn how different genes function and interact. In this thesis, both domesticated birds and laboratory mice have been used as model organisms in genetic studies of variation in pigmentation. It is hoped that work done in model organisms will be useful from a human genetics perspective, where to date, most research in the field has been limited to man and mouse. By examining other species we can gain even more knowledge of the genes related to the pigment system.

Chicken (Gallus gallus) Man started to domesticate wild animals approximately 9,000-12,000 years ago (Clutton-Brock, 1995) and it is believed that the chicken was domesti-cated in southern Asia around 8,000 years ago (West, 1988). The red jungle-fowl species has for a long time been considered the sole wild ancestor of the domesticated chicken. This was supported by genetic studies of the mito-chondrial genome (Fumihito et al., 1994; Fumihito et al., 1996). However, a recent study of domestic breeds with yellow skin showed that the beta-carotene dioxygenase 2 (BCDO2) allele in these birds originated from the grey junglefowl species (Eriksson et al., 2008). This was the first study to contradict Fumihito’s work and it remains unclear to what proportion the genome of the domestic chicken originates from the grey junglefowl.

Since the early 1900’s, extensive breeding of chickens for commercial purposes has led to two types of breeds; layers and broilers. The egg laying varieties can produce up to 300 eggs a year, in contrast to the red junglefowl, which are seasonal layers and lays around 12 eggs per clutch. The broiler has been selected for alleles favoring fast growth and large muscle mass, leading to birds weighing around four kilogram (kg), which is significantly more than the red junglefowl’s one kg (Crawford, 1990; Kerje et al., 2003a). With

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these production gains, the modern chicken has become the major source of protein globally.

The chicken has been widely used as a model organism for scientific re-search (Burt and Pourquie, 2003). It has been particularly important in the field of developmental biology, due to the relatively large and easily acces-sible embryos (Hamburger and Hamilton, 1992). The production of antibod-ies has been, and is still, of great importance in the medical industry such as was seen recently with the production of the H1N1 flu vaccine. During the course of domestication and breeding, a number of mutations affecting pig-mentation and patterning have been strongly favored by human selection (Fang et al., 2009). This genetic material, in combination with today’s pow-erful genetic tools, is a valuable resource from which to gain knowledge concerning the biology of the pigment cell.

The chicken genome was the first genome of a domesticated species to be sequenced (International Chicken Genome Sequence Consortium, 2004). It resulted in a 6.6 x coverage draft sequence of the 1x109 base pair (bp) ge-nome. In chicken, females are the heterogametic sex (Z/W) and the males are the homogametic sex (Z/Z), which is opposite to the human genome where the males are the heterogametic sex. The chicken has 38 pairs of auto-somes and one pair of sex chromosomes. Like other birds, chickens have macro- and microchromosomes. The draft genome sequence is missing much of the sequence from the microchromosomes. This is mainly due to their highly repetitive nature, making assembly of these regions problematic. In 2010 the chicken genome was resequenced from nine population pools (Rubin et al., 2010). This study revealed a number of regions under selection during domestication and improved the draft genome sequence.

There are several advantages to use chicken as a model to map genes in-volved in, for example, pigmentation. Firstly, the genome is relatively small, 1/3 the size of the human genome, yet harbors orthologs to most of the genes in man. Secondly, a large set of different colorations and patternings of pig-mentation are found in the different domestic breeds around the world. Fi-nally, establishment of crosses and linkage analysis, which will be discussed later in this thesis, can be used to map these phenotypes.

Japanese quail (Coturnix japonica) The domestication process of the Japanese quail began in Japan in the 12th century, where it was first used as a songbird and later also for meat and egg production. The Japanese empire experienced relief from tuberculosis after eating the meat, which led to an increased consumption of the bird and eggs in Japan in the latter part of the 19th century (Howes, 1964).

The quail has also been used as a model organism in science, although, not to the same extent as the chicken. Some 20-30 mutations causing pig-mentation variation have been identified (Cheng and Kimura, 1990), but

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many of the lines harboring these variants are unfortunately extinct. The quail has also been used in conjunction with the chicken in genetic research. For example, quail cells were transplanted into chicken embryos, an experi-ment which revealed that pigment cells migrate dorso-laterally in opposite to the ventrally pathway followed by the other NC stem cells (Bronner-Fraser, 1986).

Mouse (Mus musculus) The mouse is the major mammalian model organism in genetic research. It is genetically and physiologically similar to humans. The two genomes are of similar size and structure and most human genes have orthologs with the same function in the mouse. This fact is supported by mutations in ortholo-gous genes which give similar dysfunctions in the two species. There are mouse models available for most human diseases, for example, cancer (p53, Trp53), glaucoma (DBA/2J), and heart disease (Apoe) (Bult et al., 2008). Furthermore, mice are small, easy to handle and have short breeding cycles (~9 weeks). This makes it simple to set up large-scale crosses. Mouse is the only mammalian model organism in which it is reasonably easy to generate knockouts of specific genes. The knockout technique, which has revolution-ized biological science, is discussed in greater detail later in this thesis.

With these advantages, it is not surprising that the mouse is the prime model organism for coat color geneticists. To date (September 2010), 169 genes affecting pigmentation in mouse have been identified (Montoliu et al., 2010). A large number of these genes were identified as a result of mutage-nesis screens, where chemicals or irradiation have induced mutations caus-ing an easily detectable phenotype.

Forward genetics The definition of forward genetics is when you start with a phenotype of interest and move to the identification of the underlying gene or mutation. This strategy was used in Papers I and II.

Genetic variation Variation in the DNA sequence together with environmental factors, are what differentiate one individual from another. The sequence divergence in non-repetitive regions of the genome between two randomly selected human chromosomes has been estimated to 0.1% (Lander et al., 2001). If humans are compared to their closest relative, the chimpanzee, this number grows to 1.2% (Chimpanzee Sequencing and Analysis Consortium, 2005). The most common variation in the genome is single nucleotide polymorphisms

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(SNPs), where one single base has been substituted for another. Other poly-morphisms are microsatellites, insertion, deletions, and copy number varia-tions (CNVs) of duplicated sequences.

Most of this genetic variation is selectively neutral, but if the variation occurs in a DNA region of importance, e.g. a gene, it can affect gene func-tion and cause a disorder. Different kinds of genotyping techniques can be used to score genetic variation. In recent years the genotyping of SNP-sets has been widely performed, however as whole genome resequencing be-comes more affordable the popularity of this technology is increasing.

Pedigrees Many genes underlying human diseases have been identified. However, one of the drawbacks in human genetics is the need for a large set of family ma-terial to identify the gene or genes controlling a phenotype or disease. This problem is exacerbated when a phenotype appears identical, but the causal variant is not the same. However, recent genome-wide association studies have successfully been used to identify loci important for many diseases but this requires very large patient materials. The generation of large numbers of pedigreed laboratory animals such as a F2 intercross can be used to overcome the power deficiencies of the human system (Figure 3). Parental lines (P0), which are affected and unaffected by the phenotype of interest, are crossed to generate a heterozygous F1 generation. The F1 generation is intercrossed to generate a F2 generation, where the investigated phenotype will segregate. A large number of F2 animals can be generated to give power to the study and the phenotype of interest can be linked to a chromosomal region by link-age analysis (Figure 3).

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Figure 3. Overview of a three-generation intercross where the dominant phenotype Sex-linked barring is used as an example (indicated by barred chickens). Parental lines (P0) are crossed to generate a heterozygous F1, which is intercrossed to gener-ate a F2 generation. Recombination between the chromatid pairs in the F1 intercross leads to new haplotype combinations in the chromosomes passed onto the gamete. The phenotype will segregate in the F2 individuals. In this example, all individuals with at least one copy of the Sex-linked barring allele display the phenotype. A horizontal line indicates the position of the Sex-linked barring locus.

Linkage analysis Linkage analysis is a statistical method employed to spot the chromosomal location controlling a trait. It can also be used to identify the internal order of genes or genetic markers. The general approach is to look for the co-segregation of a trait and genes/markers with known location. An accurate pedigree segregating for the trait is a requirement for a linkage analysis ex-periment. Recombination between the chromatid pairs takes place during meiosis, and leads to a new haplotype in the chromosome passed onto the next generation (Figure 3). The probability of recombination between two markers located close to each other is less than for two markers that are far apart. The relative genetic distance between markers can be estimated by counting the number of recombination events between the markers of inter-est. The recombination fraction would be a direct measurement if only one recombination event could occur between two markers. However, as double, or even more recombinations, can take place between such markers, this is not the case. Multiple recombinations can be compensated by map functions.

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These functions essentially increase the estimated distance between two markers, with a greater increase occurring for larger recombination fractions. The most commonly used map functions have been described by Haldane (1922) and Kosambi (1944).

A linkage map consists of genetic markers ordered along the chromosome and the map distances between them. The distance is usually given in centi-morgan (cM), where 1cM “equals” one recombination in 100 meiosis (a 1% recombination). Linkage maps are constructed by performing linkage analy-sis on pedigrees where markers have been genotyped.

If a monogenic trait is segregating in a pedigree, it can be mapped to a chromosomal region using linkage mapping. The phenotype of interest is introduced in the same way as a genetic marker, either with a dominant or a recessive mode of inheritance. If two markers co-segregate they are in link-age with each other. A logarithm of odds (LOD) score gives the log (base 10) of the odds of the observed linkage between a phenotype and a marker, over the log of the probability that the two (marker and phenotype) are un-linked. Traditionally in linkage mapping a LOD score of greater than 3.0 has been considered significant, although the exact threshold is pedigree-specific, depending on the length of the genome and the number of tests performed.

IBD mapping and mutation detection After the localization of a chromosomal region by linkage analysis, the Iden-tical-by-Descent (IBD) mapping approach can be utilized to narrow the re-gion to be searched for the causative mutation (Figure 4). An IBD region occurs when individuals share the same ancestral haplotype on which a mu-tation first appeared. In this example, there is complete linkage disequilib-rium (LD) between the mutation and alleles at all other loci in the haplotype. Recombination will decay the LD present along the chromosome steadily during generations, but LD will endure between adjacent loci and the locus harboring the mutation under investigation in individuals carrying the pheno-type. The region in LD with the mutation can be re-sequenced in a set of individuals carrying the mutation and be compared to a set of control indi-viduals.

All positions in the IBD-region where all affected individuals share the same allele and all non-affected have another allele should be regarded as possible candidate mutations. Depending on the trait under investigation, functional experiments to reveal the causative mutation can be performed. If a regulatory mutation is suspected, a luciferase reporter assay can be set up to investigate whether the two variants have an effect on promoting a re-porter gene, or an Electrophoretic Mobility Shift Assay (EMSA) experiment can be performed to identify binding differences of transcription-factors (TF) to DNA.

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Figure 4. Identical-by-descent mapping. Suppose that a mutation (black star) ap-pears in a gene (red bar) causing barred chickens at generation 0 (g=0). At this first generation there is complete linkage disequilibrium (LD) between the mutation (B) and alleles at all other loci on this chromosome. Recombination events will decay LD gradually, but LD will persist for closely linked loci (indicated by white). This decay of LD between loci and mutation can be exploited at g=n. In this generation a minimum haplotype that is identical by descent in all individuals carrying B (yellow bar) can be identified via targeted sequencing (the figure is modified from Anders-son and Georges (2004)).

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Reverse genetics The definition of reverse genetics is when you start with a genotype, for example the generation of a transgenic mouse, and move to the identification of the phenotypic effect associated with the introduced mutation, as de-scribed in Paper III.

Generation of mouse knockouts In 2007, the Nobel Prize in physiology or medicine was awarded to Oliver Smithies, Martin J. Evans and Mario R. Capecchi for their discoveries of how to alter specific genes in mouse via the introduction of a modified DNA fragment into embryonic stem (ES) cells via homologous recombination. Their findings have made it possible to specifically knockout any gene and this has greatly aided the investigation and functional characterization of many genes. Within a few years most of the genes in the mouse genome and probably also many conserved regions will have been knocked-out (International Knockout Mouse Consortium, 2010).

Martin and Evans et al. (1974,1975) observed that in cell culture, embry-onic stem cells have the ability to differentiate into all cell types. From this discovery they saw the opportunity to use this technology for the generation of transgenic mice. In 1985, a successful experiment which inserted trans-genes into germ line mouse cells using viral RNA was published (Palmiter and Brinster, 1985). A modified gene product could be analyzed, but the integration of the modified DNA fragment was random and the host gene was unaffected. Later, Evans and coworkers used this technique to perform injections of infected embryonic stem cells into blastocysts and transferred these to a surrogate mother. Some of the subsequent mice carried the viral DNA integrated in their genomes (Robertson et al., 1986). Meanwhile, Capecchi, Smithies and co-workers had independently discovered that ho-mologous recombination occurs in mammalian cells and realized that using Evans ES-cell technique it is possible to modify every single cell in a mouse, not just in cell culture (Folger et al., 1982; Slightorn et al., 1980; Thomas et al., 1986). Smithies, Evans and Capecchi started collaborating and the Smithies laboratory showed that they could repair a mutated form of the Hprt gene in cultured ES-cells using homologous recombination (Doetschman et al., 1987). Just after, Capecchi’s laboratory inserted a neo-mycin resistance gene into the Hprt gene with the same technique, making it possible to culture the cells with this modification using antibiotics (Thomas and Capecchi, 1987). In a subsequent paper, the important protocol for posi-tive/negative selection screening was described (Mansour et al., 1988). These steps allowed for the identification of clones, which had correctly targeted genes of interest. Briefly, a neomycin resistance gene (neoR) is cloned into the targeting vector, which also has a thymidine kinase (HSV-tk)

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element adjacent to the homologous sequence. A successful homologous recombination event leads to expression of neoR, but not HSV-tk which is lost. After treatment with antibiotics only ES-cells with the correct targeting event survive.

The knockout mouse technique in use today has been refined. One exam-ple is the introduction of loxP sites into a gene of interest. When mice carry-ing these sites are mated to mice expressing the enzyme Cre-recombinase, the target DNA flanked by two loxP sites is deleted. For this kind of condi-tional knockout to be successful, it is essential that the mouse strain used expresses Cre-recombinase under a suitable promoter (Gu et al., 1994).

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Aims of the thesis

The overall aim of this thesis was to identify and characterize genes involved in the pigmentation process and subsequently to increase the understanding of how these genes act in both normal and abnormal pigmentation. The specific aims were:

• To identify the causal mutations for the pigmentation phenotypes

Silver and Sex-linked barring in chicken (Papers I and II).

• To generate and characterize a PMEL17 knockout mouse model (Paper III).

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Present investigation

Forward genetics – mapping of monogenic traits (Papers I and II) Background The phenotypes of the red junglefowl (RJF) and the domesticated White Leghorn (WL) differ considerably. WL is about two times larger and lays extensively more eggs (Crawford, 1990; Kerje et al., 2003a). The difference in plumage color is also striking. The RJF display an elegant and colorful plumage, whilst the WL has a completely white plumage. The white plum-age color of the WL has been selected for by breeders to satisfy the consum-ers desire for completely white colored skin. A great deal of research has been aimed at deciphering the genetics of plumage color. To date, five loci have been reported to explain the majority of observed phenotypes between WL and RJF; Dominant white (I), Extended black (E), Dark-brown (Db), Silver (S), and Sex-linked barring (B). Dominant white is caused by an inser-tion of 9 bp in the PMEL17 gene. This gene is important for the formation of an amyloidal structure on which the eumelanin is deposited (Kerje et al., 2004). Extended black is caused by a mutation in the coding part of the melanocortin receptor 1 (MC1R) gene, giving the bird dark black plumage (Kerje et al., 2003b). Dark-brown, was recently shown to be caused by an 8.3 kb deletion upstream of SOX10, explaining a phenotype where the ex-pression of eumelanin is reduced whereas the expression of pheomelanin is enhanced (Gunnarsson et al., submitted). The underlying genes of the Silver and Sex-linked barring loci are solute carrier family 45, member 2 (SLC45A2) and the cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) locus respectively. These findings are presented in Papers I (Gunnarsson et al., 2007) and II (Hellström et al., 2010) in this thesis.

Sex-linked barring and the gene controlling Silver were mapped in a three-generation intercross between one RJF male and three WL females. Four males and 37 females from the F1 generation were used to generate 851 F2 birds, where a variety of plumage color phenotypes segregated (Schütz et al., 2002; Kerje et al., 2003b). F2 birds were also phenotyped for numerous traits including growth and egg production (Kerje et al., 2003a). To confirm the Sex-linked barring findings in a second population, a similar intercross between RJF and White Leghorn Obese strain (OS) was used. The OS

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chickens originate from WL and is an animal model that spontaneously de-velop autoimmune thyroiditis (Cole, 1966).

The Silver locus controls the Silver (S*S) and the wild-type (S*N) pheno-types in chicken. The Silver allele was described in 1912 and was proposed to be sex-linked (Sturtevant, 1912) causing an inhibition of the red pigment of plumage. Later, a third allele at this locus, sex-linked imperfect albinism (S*AL), was identified (Cole, 1963; Werret et al., 1959). Birds homozygous for this allele have white plumage and are hatched with pink eyes, which darken with age, except for their red pupils (Mueller, 1941). In Japanese quail, the locus for sex-linked albinism (AL*A) has been shown by inter-generic crosses between male chickens and female Japanese quails to be orthologous with the Silver locus in chicken. Japanese quails expressing the sex-linked albinism phenotype lack both eumelanin and pheomelanin. An-other allele at this locus Cinnamon (AL*C), causes a dilution of the brown pigment of the plumage. Wild-type Japanese quail (AL*N) display a variable brown plumage compared to white albinos. Cinnamon birds present a diluted brown plumage pigmentation and the chicks have red eyes which again darken with age (Cheng and Kimura, 1990).

Sex-linked barring in chicken causes a band-like pattern of pigmented and non-pigmented areas of the feather (Figure 5). A barred phenotype has a camouflaging effect in nature. This is probably why it is abundant among many avian species, including the zebra finch and peregrine falcon. The phenotype is also present in many breeds of chicken, for example WL, al-though in this example the barring pattern is masked by the Dominant white locus. The Sex-linked barring locus shows a classical dosage effect, where barring homozygotes display wider white bands compared to the hetero- or hemizygotes (Figure 5B). Previous studies have shown that the white bands lack melanocytes and whilst the possible mechanism of this process has been discussed, no convincing functional data has been presented. A similar phe-notype in the chicken is autosomal barring, which also results in barred feathers but here the barring is a difference in the intensity of pigmentation rather than the distinct presence/absence of pigmentation as seen in sex-linked barring (Crawford, 1990).

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Figure 5. (A) A rooster displaying Sex-linked barring. (B) A feather from a barring homozygote (top) and a hemizygote (bottom).

In the beginning of the 20th century scientists performed tissue graft

transplantations between barred and non-barred chicks (Danforth and Foster, 1929). The aim was to see if the phenotype was affected by a factor present in the skin, such as a rhythmic variability in metabolism or hormonal secre-tions. The authors observed that skin transplanted from one sex to the other grew feathers that in structure were like those of the host and in color and barring pattern, like those of the donor.

Later, the banding pattern was proposed to be affected by autophagic de-generation of melanocytes at the proximal edge of the black band in the feather follicle (Bowers, 1988). The pattern was suggested to be caused by melanocyte death due to the excessive levels of reactive oxygen species (ROS) present during pigment production, such that when ROS levels de-crease, pigmentation progresses normally until there is again an increase in ROS levels (Bowers et al., 1994).

Color genetics is one of the most studied fields in biology, however, the underlying mechanisms of patterning in pigmentation are still poorly under-stood. Pigmentation patterning is not seen in human and mouse to the same extent as in avian species, and therefore chicken is an ideal model to use for this purpose. It is also of general biological interest to understand the con-trols of the precise on/off switch pathway underlying Sex-linked barring.

Sex-linked barring was mapped to the distal end of the q-arm of chromo-some Z in 1988 (Bitgood, 1988), and more recently it was fine mapped to a 355 kb region (Dorshorst and Ashwell, 2009). One obstacle to accurately mapping the phenotype to a chromosomal region has been the incomplete assembly of this sex chromosome. When the chicken genome was sequenced in 2004, it was from a female bird which resulted in the lower sequence cov-erage of the sex chromosomes (International Chicken Genome Sequence Consortium, 2004).

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Results and discussion Paper I – Mutations in SLC45A2 Cause Plumage Color Variation in Chicken and Japanese Quail In Paper I, we report five mutations in the SLC45A2 gene to be associated with the S*S and S*Al alleles of chicken and to the Al*A and Al*C alleles of Japanese quail. A candidate gene approach together with linkage analysis was used to map Silver in chicken to a region on the Z chromosome harbor-ing SLC45A2, and the mutations were identified after IBD mapping and re-sequencing of the coding exons. The S*S allele in all tested Silver birds, with the exception of the WL, was associated with a missense mutation changing a leucine (L) to a methionine (M) in the well-conserved transmembrane re-gion 7 (L347M; Figure 6). In all WL, the Silver allele was associated with a single nucleotide change leading to a tyrosine to a cysteine substitution af-fecting a loop region (Y277C; Figure 6). However, this change was also found in some red junglefowl birds, not supposed to carry the Silver allele, indicating that this could be a linked polymorphism. A frame-shift mutation resulting in a stop codon in exon 1 was identified as the underlying mutation in the S*Al allele (S36fs; Figure 6). The very short transcript caused by this mutation was shown to be degraded by nonsense mediated mRNA decay.

In Japanese quail, the AL*A allele carried a splice acceptor site mutation causing an in-frame skipping of exon 4 (Abn. Spl; Figure 6). Finally, an alanine to aspartic acid change in the N-terminal of the protein caused by a missense mutation in exon 1 was associated with the AL*C allele (A72D; Figure 6).

An interesting observation was that the S*S mutation causes a specific in-hibition of the expression of the red pheomelanin. A similar effect is seen in horses heterozygous for a mutation in the SLC45A2 gene. However, horses homozygous for this mutation display blue eyes and dilution of both pheo-melanin and eumelanin. No dilution effect of the eumelanin is observed in chickens homozygous for the S*S allele. This is interesting since the SLC45A2 recessive null mutation (S*Al) causes an almost full absence of both types of pigment. The Silver phenotype needs to be investigated in more detail in order to understand why mutations in SLC45A2 can have these pigment-specific effects.

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Figure 6. Membrane topology prediction of the SLC45A2 protein using TMHMM (v. 2.0). The locations of the frameshift mutation (S36fs) associated with imperfect albinism (S*AL) and the two missense mutations Y277C and L347M associated with Silver (S*S) in chicken are indicated by solid arrowheads. The A72D mutation asso-ciated with cinnamon (AL*C) in Japanese quail is marked with shaded arrowhead. The missing amino acids in the SLC45A2 protein encoded by sex-linked imperfect albinism (AL*A) in Japanese quail are shaded. Abn.Spl., aberrant splicing.

Paper II – Sex-linked barring in chickens is controlled by the CDKN2A/B tumour suppressor locus In Paper II, we demonstrate that the tumor suppressor CDKN2A/B locus controls the Sex-linked barring phenotype in chicken. This is supported by a complete association between Sex-linked barring and a 12 kb haplotype spanning the Ink4b transcript and parts of the ARF transcript, both encoded by CDKN2A/B (Figure 7A). Two non-coding SNPs in complete association with the Sex-linked barring haplotype were identified. The two SNPs, de-noted SNP1 and SNP2, are positioned in the promoter region upstream of

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ARF and in ARF intron 1, respectively (Figure 7B-C). Electrophoretic Mo-bility Shift Assay (EMSA) and luciferase reporter assays were performed to evaluate differences in transcription factor binding and transcriptional acti-vation between the alleles. No significant differences were observed, which does not necessarily imply that the SNP1 and SNP2 are of no functional importance. In these experiments mouse melanocytes were used, which of course do not mimic the biology in the feather follicle completely, as tran-scription factors expressed in chickens might not be present in the mouse cells.

Two additional SNPs were exclusively associated with Sex-linked bar-ring, however these were polymorphic between Sex-linked barring haplo-types. The two SNPs are missense mutations in the ARF transcript of CDKN2A: V9D and R10C. Both residues are well conserved among verte-brates and this part of the ARF protein is known to be functionally important (di Tommaso et al., 2009; Kim et al., 2003; Moulin et al., 2008). We denoted the Sex-linked barring allele carrying the V9D substitution B1 and the allele carrying the R10C substitution B2. Furthermore, we identified an allele, B0, identical to B1 and B2 for the 12 kb IBD region, but which lacked the V9D and the R10C missense mutations. B0 was found in one White Leghorn Line 13 bird, which is assumed to be fixed for Sex-linked barring. We can not be certain that WL Line 13 birds which carry the B0 allele also carry Sex-linked barring as the barred phenotype is masked by the expression of Dominant white.

The four SNPs associated with Sex-linked barring could be explained by the fact that one or both of the non-coding SNPs cause(s) the phenotype and that the two coding SNPs are polymorphisms with no phenotypic effect. A more plausible hypothesis is that SNP1, SNP2, or a combination of the two arose and resulted in a phenotype, and that the two missense mutations have been favored since they enhanced the phenotypic effect.

The mechanism behind this strong and precise phenotype is still unclear. Previous studies suggested that melanocyte cell death was due to high ROS levels (Bowers et al., 1994). The CDKN2A/B locus is well known to regulate cell proliferation and cell death of melanocytes, and so the hypothesis sug-gested by Bowers seems unlikely. Interesting studies of the CDKN2A/B lo-cus in mouse by Sviderskya et al. (2002), showed that the ARF knockout does not give any phenotypic effect on coat color, which supports the idea that Sex-linked barring is a gain-of-function, rather than a loss-of-function mutation.

We propose that one, or a combination, of the SNPs triggers premature cell death that in turn causes the white band of the color pattern. The forma-tion of the following black band could be explained by the recruitment of new melanocytes from a pool of stem cells, which migrate, colonize the feather follicle and produce black eumelanin. This hypothesis argues that the

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mutations identified to cause Sex-linked barring have the opposite effect in comparison to mutations causing familiar forms of melanoma in humans.

This is the first study to show that CDKN2A/B can affect coat or plumage color variation. The findings make CDKN2A/B an interesting candidate gene in other coat color studies, especially for investigations of similar pigmenta-tion patterning in zebra finches and other wild birds.

Figure 7. Identical-by-descent (IBD) mapping of Sex-linked barring. (A) Polymor-phic sites in the 12 kb region in complete association with Sex-linked barring are indicated in gray. Yellow stars indicate the positions of SNP1 and SNP2 which are closely associated with Sex-linked barring. (B) Alignment of SNP data for the IBD region. The reference sequence (line 1) and five additional individuals (line 2-6) represent birds assumed to be fixed for Sex-linked barring. SNPs are color coded with respect to the reference sequence (line 1) as follows: red, identical to the refer-ence; green, heterozygous; black, homozygous or hemizygous for the opposite allele to the reference and gray, missing data. (C) Data representing populations (lines 1-18) presumed fixed for the wild-type allele, color coded as before.

Reverse genetics – generation and characterization of a PMEL17 knockout mouse (Paper III) Background The Silver phenotype was first observed in mice as a silvering effect on coat color (Dunn and Thigpen, 1930). The gene underlying this variant was found to encode the transmembrane protein PMEL17, and was in mouse termed Silver. The orthologue in chicken is PMEL17, whereas, discussed in Paper I, the Silver gene in chicken encodes SLC45A2 and is responsible for a sepa-rate phenotype. The PMEL17 protein is exclusively expressed in eumelano-somes, the pigment producing organelle present within the melanocyte, and is thought to have a critical role in the early stages of eumelanosome bio-genesis. The PMEL17 protein is bound to the eumelanosome membrane via its transmembrane region. The protein is cleaved by a proprotein convertase, and the product forms a fibril matrix within the eumelanosome on which eumelanin is subsequently deposited (Berson et al., 2003). The function of

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the fibrils is not completely clear, but several studies suggest that they play a cytoprotective role by sequestering toxic intermediates produced during the synthesis of eumelanin (Fowler et al., 2006). The fibril organization mimics the amyloid fibrils found in Alzheimer’s and Parkinson’s disease patients and are the only non-pathological amyloid structures with a known function (Kelly and Balch, 2003).

PMEL17 is well conserved between species, indicating an important function, and mutations in the Silver gene have been reported to dilute the black or brown pigment (eumelanin) in mouse (Dunn and Thigpen, 1930; Kwon et al., 1991; Martinez-Esparza et al., 2000), chicken (Kerje et al., 2004), horse (Brunberg et al., 2006), zebrafish (Schonthaler et al., 2005), cattle (Kuhn and Weikard, 2007), and dog (Clark et al., 2006). However, no complete loss-of-function in a mammal has been reported. The first reported mutation affecting PMEL17 function showed that the recessive Silver allele in mouse is caused by a single bp insertion that truncates the last 25 C-terminal residues of PMEL17 within the cytoplasmic domain (Martinez-Esparza et al., 2000; Solano et al., 2000). In this case hair become less pig-mented on black backgrounds and appears gray over time (Dunn and Thig-pen, 1930; Kwon et al., 1991). The graying phenotype was more pronounced in Silver mice heterozygous at the Tyrosinase-related protein locus (Tyrp1 or B) compared to Silver mice homozygous B/B or b/b (Silvers, 1979). Mi-croscopic examination of these mice revealed their eumelanosomes to be larger and rounder when compared to the wild-type (Theos et al., 2006). It is surprising that only one mutation has been identified in the Silver gene in mouse. Mutations in genes regulating pigmentation are easily detected in mutagenesis screens. To date (September, 2010), 169 genes affecting pig-mentation have been identified and in many of these genes several alleles have been reported. The fact that only one mutation in Silver has been identi-fied in mouse indicates either (i) that PMEL17 has other yet unknown func-tions and that inactivating mutations are embryonic lethal, or (ii) that com-plete loss-of-function mutations at the Silver locus have no or only mild phenotypic effects that are not readily picked up in extensive mouse screens.

Results and discussion Paper III – Inactivation of the Silver gene alters the shape of eumelanosomes but has only a subtle effect on pigmentation In Paper III, we generated a knockout mouse line in which exons 2-3 of the Silver gene are deleted. The knockout mice are viable, fertile, and show no obvious developmental defects. The Silver transcript in the knockout showed a 279-fold down regulation compared to the wild-type. Primary cultures of skin-derived melanocytes were established from wild-type and Silver-/- mice. These cells were used for immunoblotting, immunoflourescence microscopy,

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bright-field microscopy and electron microscopy experiments. The absence of PMEL17 protein in our Silver-/- primary melanocytes was confirmed by immunoblotting experiments, where these cells were not immunoreactive to any of the PMEL17-specific antibodies, but expressed normal levels of two other pigment-cell specific proteins, Tyr and Tyrp1. No obvious alteration in the degree of pigmentation could be observed in Silver-/- primary melano-cytes compared to the wild-type under the bright-field microscope. This contradicts the general thought that PMEL17 is a key player in the synthesis of pigment. Bright-field microscopy at higher magnification (100x) clearly demonstrated an altered shape of the eumelanosomes (spherical instead of rod shaped) in the knockout mice in both the uveal melanocytes of the chor-oid layer as well as in RPE. This demonstrates that PMEL17 is required for normal development of the eumelanosomes in both these cell types. The electron microscopy experiment of cultured melanocytes from the knockout mice convincingly confirmed that the eumelanosomes are spherical in con-trast to the cigar-like shape seen in the wild-type (Figure 8). However, they are not enlarged as previously reported in the spontaneously mutated silver mice (Theos et al., 2006). This finding confirms the PMEL17-fibrils to be essential in giving eumelanosomes their elongated form.

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Figure 8. Electron microscopy. The eumelanosomes in the Silver+/+ melanocytes are rod shaped, whilst the eumelanosomes in the Silver-/- melanocytes are round.

Melanin has an important role in the skin, the eye and the inner ear. Skin

sections from Silver+/+ mice clearly showed PMEL17 immunoreactive cells in the hair follicles. These were not observed in the Silver-/- homozygotes. However, Silver-/- mice did display normal eye pigmentation, with melanin being present in both the RPE and in uveal melanocytes located in the chor-oid layer of the eye. No PMEL17 expression was observed in the uveal melanocytes, but expression was possibly detected in RPE by immunohisto-chemistry. At present we cannot exclude the possibility that the immunore-activity observed in the RPE represents a non-specific signal.

The Silver-/- mice did not display any striking effect on pigmentation on a C57BL/6 background. We therefore set up a three-generation intercross be-tween Silver-/- mice (backcrossed onto C57BL/6 background) and BALB/C mice. In addition to the segregation at the Silver locus, this intercross segre-gates for three other coat color loci: agouti-signaling protein (Asip), i.e. the classical Agouti (A) locus; tyrosinase-related protein 1 (Tyrp1), i.e. the clas-sical Brown (B) locus; and Tyrosinase (Tyr), i.e. the classical Albino (C) locus. The Silver-/- mice showed a subtle effect on black, brown, agouti, and

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brown agouti backgrounds (Figure 9). On a black background, a weak silver-ing effect was seen and the coat color was diluted compared to wild-type. In addition, a reduced pigmentation on paws and tail was evident (Figure 9e-f). The most pronounced effect of the Silver knockout was observed on brown background, where the brown pigmentation was markedly diluted (Figure 9b). Brown agouti, Silver-/- mice had an apparent dilution of the dark hairs, whilst the yellow pigmentation seemed unaffected (Figure 9c). The most subtle effect of the Silver-/- genotype was noted in agouti-colored mice, but their paler tails distinguished the knockouts from the wild-type litter mates (Figure 9d).

Figure 9. Pigmentation phenotypic-effects of the Silver-/- genotype on four different genetic backgrounds of an F2 intercross. Silver-/- (left) and Silver+/+ (right) mice are shown for each group. (a) Non-agouti black; the black Silver-/- mice display a subtle dilution of coat color whilst the skin of tail and feet are considerable less pigmented compared with the wild-type (e-f). (b) Brown; the strongest dilution effect of the PMEL17 inactivation on coat color pigmentation are seen in brown mice. The pig-mentation of the skin is also significantly reduced on the brown background. (c) Brown agouti; the black hairs of the Silver-/- mice on brown agouti background is diluted, giving the coat a more yellow/light brown appearance compared to the wild-type. (d) Agouti; the weakest phenotypic effect associated with the Silver-/- genotype is seen in the agouti mice. The coat and skin color is slightly lighter in relation to the wild-type.

This project was initiated to explain why only a single mutant allele has

been identified at the Silver locus in mice despite the extensive screening for pigmentation mutations that have been carried out in this species, and the fact that mutations at this locus are common in domestic animals (chicken, dog, horse and cattle). We proposed two explanations, (i) that PMEL17 has other yet unknown functions and inactivating mutations are embryonic le-thal, or (ii) that complete loss-of-function mutations at the Silver locus have

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no or only mild phenotypic effects that are not readily picked up in extensive mouse screens. Our study has conclusively demonstrated that the latter ex-planation is correct and this is consistent with the view that PMEL17 is a melanocyte-specific protein. It is surprising that point mutations in the Silver gene in several species cause strong dilution of the eumelanin, whilst the effect in our knockout is moderate. This shows that dominant-negative al-leles can have more severe effects on the phenotype than null alleles at this locus. Amyloid structure formation is known to cause disease, and if the amyloid structure formed by PMEL17 is expressed at a location other than the eumelanosome it may be detrimental. Hence, the mild effect of the knockout could be explained by the fact that the absence of PMEL17 does not lead to formation of any harmful amyloid structure and that PMEL17 is not absolutely required for normal pigmentation. Our knockout mice will be an excellent tool to further investigate the function of PMEL17 in vivo.

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General discussion and further perspectives

The results presented in this thesis reveal a number of mutations causal for the plumage color and patterning variation found in chicken and Japanese quail. In addition, a Silver knockout mouse model has been generated and analyzed. The data from all three papers provides material for further re-search.

In Paper I we report five mutations in SLC45A2 causing alterations to the pigmentation of the plumage in both chicken and Japanese quail. The major discovery in this paper is the specific inhibition of pheomelanin in Silver chickens, whilst null mutations at this locus cause an almost complete ab-sence of both pheomelanin and eumelanin. This cannot be explained by the current understanding that the main function of the SLC45A2 protein is to transport TYR into the premelanosomes, since TYR is even more vital for the production of eumelanin. The seven transmembrane SLC45A2 transport protein forms a pore, but it is still unclear which molecules the protein trans-ports and through which membrane the molecules are transported. This has to be investigated in more detail before the full mechanism of the protein is understood. From a genetics perspective, there are likely more mutations to pinpoint in the SLC45A2 gene. Sex-linked albinism is assumed to be present in turkey, budgerigar, and canary birds (Smyth, 1990) and SLC45A2 is obvi-ously a good candidate gene in these.

In Paper II we show that Sex-linked barring in chickens is controlled by the CDKN2A/B tumor suppressor locus. The locus encodes two proteins, INK4B and ARF. The genetic analysis indicates that missense mutations in ARF or mutations in the promoter region of the ARF transcript are causing Sex-linked barring. However, this has yet to be confirmed by functional studies, such as measuring possible expression differences of the two tran-scripts in feather follicles from Sex-linked barring and wild-type birds. The B0 haplotype found in one bird assumed to carry Sex-linked barring masked by the Dominant white allele will be further investigated. The B0 haplotype is identical in the 12 kb IBD region to the B1 and B2 haplotypes, except that it has neither the V9D nor the R10C missense mutation. We will design breeding experiments to investigate whether the B0 haplotype causes Sex-linked barring or not. This will clarify if the phenotype is caused by the mis-sense mutations or non-coding SNP1 and SNP2. Additionally, we have a general interest in revealing the functional impact of the V9D and R10C substitutions and how they may affect the ARF protein. In both human and

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mouse, ARF is part of the ARF-p53-MDM2 (mouse double minute 2 ho-molog) network, where it either regulates the cell cycle and transition from G1 to S-phase, or directs the cell cycle to apoptosis. Chicken ARF shares many properties with mammalian ARF. Chicken ARF has been shown to coimmunoprecipitate with MDM2 just like human ARF and also protect p53 from MDM2-mediated destruction (Kim et al., 2003). We are therefore in-terested to set up experiments with expression vectors harboring wild-type ARF, V9D ARF and R10C ARF. These will be co-transfected with a lu-ciferase vector with a p53 promoter. In order to investigate if either of the two mutations has an effect in promoting p53-expression.

In Paper III we generated and characterized a Silver knockout mouse. We identified that PMEL17 is not essential for pigment synthesis. The eumela-nosomes within the melanocytes of the Silver-/- mice are round in contrast to the cigar-like shape present in wild-type animals. This feature was docu-mented in primary cultures from skin-derived melanocytes as well as in reti-nal pigment epithelium cells and uveal melanocytes. The altered shape of the melanosomes in the eye of the Silver-/- mice may affect vision and could possibly cause age-related eye disorders. Studying this in more detail and revealing the long-term effect of this phenotype may be of clinical impor-tance, since Silver-/- humans are likely to have a similar eye phenotype. To discern whether the IHC staining of RPE from Silver-/- mice is specific or artefactual, additional antibodies will be used. The Bruch’s membrane of RPE tends to give background staining and could explain the unexpected signal of the RPE. Furthermore, it would be interesting to investigate the auditory capacity of the Silver-/- mice, given melanocytes are essential for hearing but melanin is not. The cellular role of melanocytes in the ear is under debate, but it could be possible that the amyloid structure formed by PMEL17 is required for normal function. Audition is one of the more than 300 phenotypic measures that will be performed on Silver-/- mice at the Ger-man Mouse Clinic. The comparison of these extensive results to wild type mice will go a long way to revealing the full phenotypic extent of the PMEL17 knockout.

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Svensk sammanfattning

I denna avhandling har gener som reglerar pigmentcellens biologi studerats. Färgegenskaper, såsom pälsfärg och pälsteckning i olika arter, har använts av genetiker som modell för Mendelsk genetik i över 100 år. Eftersom dessa egenskaper är relativt lätta att identifiera är de värdefulla modeller för att förstå hur gener som styr pigmentering fungerar och interagerar. Pigmentcel-lens utveckling verkar till stor del ske på samma sätt och styras av samma gener i de flesta arter. Därför kan vi dra nytta av upptäckter i modellsystem såsom höns och möss för att förstå hur normal pigmentering i människa går till men också för att förstå vad som går fel vid sjukdomar relaterade till pigmentcellen som t ex hudcancer.

I det första delarbetet visar vi att färgvarianter hos domesticerade höns och vaktel orsakas av mutationer i SLC45A2-genen som kodar för ett trans-membranprotein. En fullständig utslagning av denna gen orsakar en färgva-riant som kallas ofullständig albinism medan missense-mutationer (påverkar proteinets aminosyrasekvens) är associerade med avsaknad av det röda pig-mentet hos höns, vilket kallas Silver-pigmentering. Flera studier på andra arter, inklusive människa, har visat att normal SLC45A2-funktion krävs för normal utveckling av pigmentering, men dess fullständiga funktion är fortfa-rande oklar. Den mest intressanta upptäckten i vår studie var att vi kunde identifiera mutationer som har en specifik effekt på utvecklingen av rött pigment (feomelanin) men som inte påverkar det svarta pigmentet (eumela-nin).

I det andra delarbetet studerades Sex-linked barring i höns. Denna färgva-riant ger hönsens fjädrar ett zebraliknande mönster. När fjädern växer ut inhiberas och aktiveras pigmenteringen cykliskt vilket resulterar i en randig fjäder. Den här typen av fjädrar är mycket vanliga hos vilda arter eftersom det ger ett kamouflerande skydd. Vi fann att mutationer i tumörsuppressor-genen CDKN2A som kodar för ARF-proteinet orsakar Sex-linked barring i höns. ARF-proteinet har en central roll för cellcykelreglering och påverkar därmed hur fort celler delar sig. Mutationer som inaktiverar CDKN2A är den vanligaste orsaken till ärftliga former av melanom hos människa. Vår hypo-tes är att mutationen hos höns har en motsatt effekt och orsakar för tidig celldöd snarare än tumörer. Detta är första gången mutationer i CDKN2A visat sig påverka pigmentering hos någon art.

I det tredje delarbetet genererades och karaktäriserades en knockoutmus, där Silver-genen, som i mus kodar för PMEL17-proteinet, slogs ut. Detta

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protein är uttryckt i de pigmentproducerande-strukturerna eumelanosomer som återfinns i melanocyter och har antagits ha en kritisk roll för produktio-nen av svart pigment. Spontant uppkomna mutationer i denna gen hos mus, höns, hund, häst, nötkreatur och zebrafisk orsakar pigmentdefekter. I vår studie visar vi att avsaknad av PMEL17-proteinet förändrar den karakteris-tiska cigarr-formade eumelanosomen till en helt sfärisk struktur. Avsaknaden av proteinet påverkar dock intensiteten av pigmenteringen mindre än de tidi-gare beskrivna spontant uppkomna mutationerna. Detta tyder på att dessa mutationer har en dominant-negativ effekt på eumelanosomens utveckling som är mer drastisk än en total avsaknad av proteinet. Dessa resultat ger ny grundläggande kunskap om PMEL17-proteinets betydelse för pigmentcel-lens funktion.

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Acknowledgements

Studies presented in this thesis were performed at the Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden.

I would like to thank the following people: Leif Andersson, my main supervisor, for educating me in a successful and stimulating scientific environment. It has been encouraging investigating science with you. Your genuine optimism, passion for science and lack of stress hormones are impressive. Thank you!

Susanne Kerje, my co-supervisor, for valuable discussions regarding the pigment cell, letters of recommendation, feedback on manuscripts, and many tedious hours of mice handling.

Klas Kullander, my co-supervisor, for technical advice in the design of the knockout targeting construct. All co-authors on the papers, past and present members of the lab.

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Figure 10. Schematic of additional people I would like to acknowledge (indicated in white boxes) for different activities (indicated by icons). Missing people are wel-come to fill in their name into the empty box and a suitable activity into the empty icon.

44

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