Characterization of CFAP43 and its function in motile cilia

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leerzeile

Medizinische Hochschule Hannover leerzeile

Institut für Molekularbiologie

Characterization of CFAP43 and its function in motile cilia

INAUGURALDISSERTATION

zur Erlangung des Grades einer Doktorin der Naturwissenschaften - Doctor rerum naturalium -

(Dr. rer. nat.)

vorgelegt von leerzeile Ev Rachev

leerzeile aus Wolmirstedt

leerzeile leerzeile Hannover 2017

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Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover leerzeile

Präsident: Prof. Dr. med. Christian Baum

Wissenschaftliche Betreuung: Prof. Dr. rer. nat. Achim Gossler

Wissenschaftliche Zweitbetreuung: Prof. Dr. rer. nat. Dietmar Manstein

1. Referent: Prof. Dr. rer. nat. Achim Gossler 2. Referent: Prof. Dr. rer. nat. Dietmar Manstein 3. Referent: Prof. Dr. med. Matthias Ochs leerzeile

Tag der mündlichen Prüfung: 17.11.2017

Prüfungsausschuss:

Vorsitz: Prof. Dr. rer. nat. Achim Gossler 1. Prüfer: Prof. Dr. rer. nat. Achim Gossler 2. Prüfer: Prof. Dr. rer. nat. Dietmar Manstein 3. Prüfer: Prof. Dr. med. Matthias Ochs

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Zusammenfassung

Ev Rachev

Charakterisierung von CFAP43 und seinen Funktionen in motilen Zilien

Ursprünglich wurdeCfap43in einer Gruppe von Microarrays als ein FOXJ1 Zielfaktor identifiziert, dessen Expression mit motilen Zilien in der Lunge assoziiert ist. Zudem wurde gezeigt, dass CFAP43 überwiegend in Geweben exprimiert ist, welche motile Zilien en- thalten (Doktorarbeit Michaela Mai, 2012). Innerhalb der vorliegenden Doktorarbeit konnten vorangegangene Expressionsstudien bestätigt werden, und darüber hinaus wurde nicht nur die Lokalisierung von CFAP43 in motilen Zilien nachgewiesen, sondern auch seine Notwendigkeit für die Funktion von einigen Typen motiler Zilien. In vier Teilprojekten wurden 1. Antikörper gegen CFAP43 hergestellt und charakterisiert, 2. Eigenschaften und Funktionen von CFAP43 in Zellkultur untersucht, und seine in vivo Funktion wurde 3. im Zebrafisch- und 4. im Maus-Model analysiert.

Um die Lokalisierung von CFAP43 detailliert untersuchen zu können, wurden monoklonale Antikörper generiert und gemeinsam mit bereits vorhandenen polyklonalen Antikör- pern charakterisiert. Einige Antikörper wurden identifiziert, die verschiedene Epitope von CFAP43 im Western Blot und in der Immunpräzipitation detektieren. Zudem wurden Be- dingungen etabliert, unter denen monoklonale Antikörper endogenes CFAP43 in Immun- fluoreszenz-Färbungen markieren.

In vitro wurden zunächst Instrumente entwickelt, mit denen das Expressionslevel von CFAP43 in der Zellkultur verändert werden konnte. CFAP43 Reduktion wurde durch siR- NAs und shRNAs erreicht, wobei letztere durch lentivirale Expressionssysteme in Zellen übertragen wurden. Überexpressions-Phänotypen, die zuvor beobachtet wurden (Mai, 2012), konnten durch induzierbare CFAP43 Überexpression in einer murinen Zelllinie nicht bestätigt werden. Zudem wurde die primäre Kultur von murinen trachealen Epithelzellen als Mod- ellsystem für die Entstehung mutipler motiler Zilien etabliert, um hierin eine mögliche Rolle von CFAP43 in der Biogenese von motilien Zilien zu untersuchen. Der Knock-down von CFAP43 in isolierten trachealen Epithelzellen führte dazu, dass in diesem System in vitro weniger multiziliierte Zellen ausgebildet wurden, obwohl das Ziliogenese-Programm ges- tartet wurde. Neben einer Analyse der möglichen Funktionen von CFAP43 wurden auch po- tentielle Interaktionen in Zellkultur untersucht. Mögliche Interaktionspartner von CFAP43 wurden mit Hilfe verschiedener Screens identifiziert und im Anschluss mithilfe von Ko- Immunopräzipitationen validiert. Hieraus resultierte eine heterogene Gruppe von Proteinen,

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von Zilien assoziiert wurde. Aufgrund des Wissens, dass IQCB1 für den selektiven Transport von Proteinen in das Zilium benötigt wird, erscheint es jedoch wahrscheinlicher, dass der Kontakt zwischen CFAP43 und IQCB1 bei dem Eintritt von CFAP43 in das Zilium entsteht, als dass beide Proteine eine gemeinsame Funktion im Zilium erfüllen.

Im Gegensatz zur Maus undXenopus Embryonen konnte keine spezifische Lokalisierung derCfap43Expression in Zellen mit motilen Zilien in Zebrafisch Embryonen festgestellt werden. Ungeachtet dessen führt der Knock-down von CFAP43 zu Phänotypen, die im Ze- brafisch bereits mit der Fehlfunktion von Zilien assoziiert wurden. Namentlich resultierte der Knock-down mittels zweier verschiedener Morpholinos in einer ventral gekrümmten Körper- achse, perikardialen Ödemen, Hydrocephali, Fehlbildungen der Otolithen und erweiterten pronephrischen Gängen. Diese Phänotypen konnten durch die Injektion von murinerCfap43 mRNA zum Teil gerettet werden. Dies zeigt zum einen die Spezifität der Morpholinos und zum anderen, dass die Funktion der beiden Proteine evolutionär konserviert ist.

Um die Funktion von CFAP43 für motilie Zilien in Säugetieren und den daraus resultieren- den Einfluss auf die Entwicklung zu untersuchen, wurde eine Knock-out Maus generiert.

Homozygote Tiere waren lebensfähig und erschienen zumeist gesund. Nur in wenigen Fällen kam es zur Bildung eines äußerlich sichtbaren Hydrocephalus. Durch die Präparierung der Gehirne wurden allerdings erweiterte laterale Ventrikel in jeder adulten Knock-out Maus nachgewiesen. Zusätzlich akkumulierten die Tiere Mukus in den Nasennebenhöhlen, was auf eine Fehlfunktion der motilen Zilien in den Atemwegen hinweist. Außerdem waren die Männchen infolge von schwere Fehlbildungen der Spermien infertil. Diese Form der Missbildungen von Spermien wurde schon zuvor unter dem Namen "Dysplasia of the fibrous sheath" beschrieben und umfasst stark verkürzte und aufgerollte Flagellen sowie ge- spaltene Axoneme. Das Fibrous Sheath ist in diesen Spermien falsch lokalisiert oder nicht vorhanden. Elektronenmikroskopische Untersuchungen von Mausspermien von Tang et al.

(2017) sowie im Rahmen dieser Doktorarbeit zeigten, dass die axonemale Struktur in Sper- mien von Knock-out Mäusen vollständig aufgelöst ist. Molekulare Prozesse, welche den beobachteten Phänotypen zugrunde liegen, müssen weiter untersucht werden, um die Funk- tion von CFAP43 im Detail zu verstehen. Im Gesamten betrachtet wurde mit der Cfap43 Knock-out Maus ein Modellsystem entwickelt, welches Einblick in zwei Erbkrankheiten gibt, die mit motilen Zilien assoziiert sind. Diese sind zum einen die primäre ziliäre Dyskinesie und zum anderen Dysplasia of the fibrous sheath. Beide Krankheiten können abhängig vom genetischen Hintergrund getrennt voneinander oder auch gemeinsam auftreten.

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Abstract

Ev Rachev

Characterization of CFAP43 and its function in motile cilia

Cfap43 was initially identified in a set of microarrays as a FOXJ1 effector, whose expression correlates with the formation of motile cilia of the lung. In addition its expression in tissues containing motile cilia has been shown (thesis Michaela Mai, 2012). During the work presented here, previous expression studies could be substantiated. Furthermore CFAP43 protein was shown to localize to motile cilia in the mouse, and to be essential for function of motile cilia in some tissues. Within four subprojects 1. antibodies against CFAP43 were generated and characterized, 2. CFAP43 properties and functions were investigated in cell culture, and itsin vivofunction was studied 3. in zebrafish and 4. in mouse models.

To analyze CFAP43 localization monoclonal antibodies were produced and evaluated together with already existing polyclonal antibodies. Several antibodies directed against different epitopes were found to bind to CFAP43 in western blot and immunoprecipitation.

Additionally, conditions were established that allowed for detection CFAP43 in immunofluorescence staining using monoclonal antibodies.

Forin vitroexperiments tools were developed to manipulate CFAP43 levels in cell culture.

Knock-down was established using siRNAs and shRNAs, which were transduced using a lentiviral expression system. Overexpression phenotypes, which had been proposed before (Mai, 2012), could be excluded using a murine cell line allowing for inducible expression of CFAP43. Additionally, primary cell cultures of murine tracheal epithel cells were established and used to investigate a potential role of CFAP43 during the generation of multiple motile cilia in these cells. Fewer multiciliated cells were present after knock-down of CFAP43, indicating contribution of CFAP43 to cilia formation by an as yet undefined mechanism. In addition to functional analysis of CFAP43 in cultured cells, putative interaction partners were evaluated to identify a potential process, which might require CFAP43. Potential interaction partners were determined using yeast-two-hybrid screens and affinity purification of CFAP43 containing complexes with their subsequent mass spectrometric analysis. Puta- tive interaction partners were validated by co-immunoprecipitation. This analysis resulted in a heterogenous group of proteins, of which only one, IQCB1, which localizes to the basal body, has been associated with ciliogenesis before. However, current knowledge regarding the function of IQCB1 in regulation of selective entry of proteins into the cilium suggests that this interaction might occur during transport of CFAP43 into the cilia, rather than providing

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In contrast to the mouse and Xenopus embryos, no specific expression of Cfap43 in cells carrying motile cilia could be determined in zebrafish embryos. Nevertheless knock-down and rescue studies revealed that CFAP43 is linked to motile cilia function in the fish. Upon knock-down using two independent morpholinos the embryos developed defects, which have been linked to cilia function before, namely ventral body axis curvature, pericardial edema, hydrocephali, otolith malformations and dilated pronephric ducts. These phenotypes were partially rescued when murine CFAP43 was expressed in morphants, demonstrating both specificity of the morpholinos and evolutionary conservation of the function of fish and mouse CFAP43.

To understand the function of CFAP43 for function of motile cilia in mammals as well as its contribution to normal development, a knock-out mouse was generated. Homozygous knock-out mice were viable and most animals appeared externally normal. Few mice developed an externally visible hydrocephalus, but upon histological analysis of brains from adult mice dilated lateral ventricles were observes in all individuals. Additionally, mucus accumulations were found in nasal cavities of knock-out mice, pointing towards defective cilia motility in the airways. Another observed phenotype was male infertility caused by sperm malformations that have been described before and are named "dysplasia of the fibrous sheath". The hallmark of malformed sperm cells are strongly shortened tails, whose principal piece is often coiled or contains split axonemes. The fibrous sheath in these spermatozoa appears to be dislocated. Additionally, in a recent independent study the central microtubule pair of sperm flagella was shown to be absent in sperm from sterile patients carrying mutations inCFAP43 (Tang et al., 2017). Independent electron microscopy studies performed by Tang and colleagues and as a part of this thesis showed the axonemal structure to be completely disrupted in sperm fromCfap43knock-out mice. Molecular processes requiring CFAP43 still need to be investigated for detailed understanding of CFAP43 function in motile cilia. Hence, with the Cfap43 knock-out mouse a new mouse model became available, that might provide insights into the mechanisms, which lead to primary ciliary diskinesia and dysplasia of the fibrous sheath, two genetically inherited diseases, which are caused by defects of motile cilia and can, depending on the genetic background, occur independently or be interconnected.

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1 Introduction

In the recent decades defects in cilia structure and function turned out to be the cause of a group of genetically inherited diseases collectively referred to as ciliopathies (reviewed in Gerdes et al., 2009). Primary ciliary dyskinesia (PCD) is a subgroup of these diseases, which is the consequence of defects in cilia motility. For better understanding of these diseases and potential improvements of medical treatment in the long term many efforts have been un- dertaken to understand biogenesis and function of cilia. However, the knowledge regarding cilia components and their function is still incomplete. Therefore the analysis and functional characterization of novel cilia components such as CFAP43 is essential.

CFAP43 was identified in screens for identification of FOXJ1 target genes (Mai, 2012;

Stauber et al., 2017). FOXJ1 is a key regulator of biogenesis of motile cilia (Alten et al., 2012; Brody et al., 2000; reviewed in Choksi et al., 2014b). Cfap43is a FOXJ1 target gene and its analysis especially with regard to motile cilia is the subject of this thesis. In addition mutations in CFAP43were recently identified to be the cause of male sterility in humans (Tang et al., 2017). For better understanding of the experiments and their results a brief introduction will be given concerning ciliary structure and function in general and the special features of different kinds of motile cilia. Additionally the screen, in whichCfap43was identified, will be explained as well as results from previous investigations regarding CFAP43.

1.1 General structure and function of cilia

Cilia are microtubule-based structures, which project from the surface of cells. Detailed investigation regarding the ciliary structure were performed in primary cilia, which are non- motile and transiently project from the surface of cycling cells. Therefore, the general ciliary structure is shown using a primary cilium as example (figure 1.1 A), whereas additional features of other types of cilia will be explained later. The base of primary cilia is formed by a pair of centrioles, which can be discriminated by distal and subdistal appendages that appear only at the mother, but not at the daughter centriole (Paintrand et al., 1992). The mother centriole forms the basal body, which is anchored to the cell membrane. Since the centrosome is needed for proper chromosome segregation during cell division, primary cilia exist only in the G0 phase of quiescent cells or during the G1 phase of proliferating cells and are disassembled before re-entry of the cell cycle. Centrioles are build of nine microtubule triplets, which are arranged in a cartwheel formation. The transition zone is positioned at the base of the ciliary axoneme, directly above the basal body, with the distal appendages of the basal body forming the transition fibers (Sorokin, 1968). It is thought to act as a diffusion

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Figure 1.1: Structure and types of cilia. AThe ciliary axoneme protrudes from the basal body, which is formed by the mother centriole. The microtubular triplets of the basal body are continued as doublets in the cilium (modified from Ke and Yang, 2014).BCilia can be divided in several different types. Some of them are the sperm flagellum, motile and sensory monocilia at the left-right organizer, mutliple motile cilia at epithelia of the airways, oviduct and ventricles and primary cilia which can act as signaling or sensory cilia (from Choksi et al., 2014b).

barrier and to control the trafficking of proteins from and to the cilium together with the BBSome (Barbelanne et al., 2015; Jin et al., 2010; Nachury et al., 2007; reviewed in Garcia- Gonzalo and Reiter, 2017). The ciliary axoneme consists of nine microtubule doublets, which protrude from the basal body. The cilium is build by intraflagellar transport (IFT) from the microtubule minus end, which is docked to the microtubule triplets of the basal body, to the plus end forming the ciliary tip. IFT is divided into anterograde transport, which is needed for axoneme elongation, and retrograde transport for trafficking of proteins from the tip to the base, e.g. for recycling of IFT components and cilia disassembly. In anterograde transport kinesin-2 carries the IFT complex B and its cargos from the ciliary base to the tip (Cole et al., 1998). In retrograde transport dynein-2 moves the IFT complex A together with its cargos back to the base (Pazour et al., 1998). In cases of defective anterograde transport no cilia can be formed, whereas disturbed retrograde transport leads to shortened cilia that display bulbs containing IFT particles along their length (Huangfu and Anderson, 2005; Huangfu et al., 2003; Pazour et al., 1998, 1999).

Cilia can be divided in several classes (figure 1.1 B). The most simple way is the distinction into non-motile and motile cilia. Non-motile cilia comprise primary cilia, which function mainly in intracellular signaling and mechanosensation (e.g. mechanosensory cilia within the kidney tubules measuring urine flow), and highly specialized sensory cilia, for example the connecting cilium in photoreceptor cells and sensory cilia of olfactory neurons. Classically

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1.2 Transcriptional regulation of motile ciliogenesis

non-motile cilia have a 9+0 conformation, in which the axoneme consists of nine microtubular doublets without a central pair of microtubuli. An exception are the sensory cilia of olfactory neurons, which contain the central pair (reviewed in Choksi et al., 2014b). In contrast the most motile cilia display a 9+2 conformation. Here, the central pair is connected to the nine outer doublets by radial spokes (Afzelius, 1959; Gibbons and Grimstone, 1960). Other features, which are not present in primary cilia, are nexin bridges connecting the microtubule doublets (Gibbons, 1963) as well as inner and outer dynein arms. Inner dynein arms (IDA) control wave formation along the axoneme and sliding of outer dynein arms (ODA) results in bending of the cilia (Brokaw and Kamiya, 1987; Elam et al., 2009). Motile monocilia in the left-right organizer, also called organ of laterality, determine left-right asymmetry in vertebrates and a long specialized cilium, the flagellum, facilitates sperm movement (Nonaka et al., 1998). Multiple motile cilia are found on the apical surfaces of epithelia in the brain ventricles, oviducts and airways.

Biogenesis of multiple cilia is a special challenge for the cell, because a few hundred basal bodies must be generated from one existing centrosome. Centriole amplification happens both, in a mother-centriole dependent pathway and usingde novoformed nucleation centers called deuterosomes (Dehring et al., 2013; Zhao et al., 2013). Deuterosomes are formed at the daughter centriole, a process which is controlled by CCNO by a so far unknown mechanism (Funk et al., 2015; Jord et al., 2014). To start centriole biogenesis CEP63 is recruited to the mother centriole, whereas its paralouge DEUP1 binds the deuterosome (Zhao et al., 2013). Subsequently CEP152 and PLK4 bind these proteins and recruit SASS6 for cartwheel formation, which then allows downstream centriole assembly (Dehring et al., 2013; Hatch et al., 2010; Nakazawa et al., 2007; van Breugel et al., 2011). Fully assembled centrioles leave their place of formation and relocate to the apical surface of the cell, where they become connected to the apical actin web (Gomperts et al., 2004; Zhao et al., 2013). After docking of basal bodies to the actin web ciliogenesis takes place (Gomperts et al., 2004; Pan et al., 2007).

1.2 Transcriptional regulation of motile ciliogenesis

The biogenesis of motile cilia is regulated by a complex network of transcription factors that is best studied for the multiciliated cells of the airways and of theXenopusepithelium. Two early acting determinants of the multiciliated cell fate are the transcription factors Multicilin and GEMC1, which were shown to activate expression of the transcription factor FOXJ1 and and transcriptional activator MYB. In this cascade GEMC1 was shown to act upstream of Multicilin. The motile ciliogenesis program is inhibited by Notch signaling via inhibition of GEMC1 (Kyrousi et al., 2015; Zhou et al., 2015). Multicilin was recently shown to form a

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ternary complex with the transcription factors DP1 and E2f4 or E2f5 to activate expression of its target genes (Ma et al., 2014). The Multicilin target p73 is a member of the p53 protein family, which directly activates expression of the transcription factorsMyb,Foxj1, Rfx2and Rfx3as well as other genes involved in ciliogenesis in murine tracheal epithel cells (mTECs) (Marshall et al., 2016; Nemajerova et al., 2016). Furthermore NOTO is required for expression ofFoxj1in the node, which is the murine left-right organizer (Alten et al., 2012).

MYB facilitates centriole amplification, which is a crucial step during multiciliogenesis, since hundreds of basal bodies emerge from one centrosome. Additionally MYB induces expression of FOXJ1 early in ciliogenesis (Tan et al., 2013). FOXJ1 activates a transcriptional program, which is needed for migration and apical docking of basal bodies and subsequent steps of ciliogenesis. Generation of multiple basal bodies does not require FOXJ1 (Brody et al., 2000; Gomperts et al., 2004; Pan et al., 2007). Additionally some RFX factors are needed for ciliogenesis, with RFX2 and RFX3 being essential for motile cilia in some contexts (reviewed in Choksi et al., 2014b). The latter two could be shown to co-activate FOXJ1 by stabilization of the binding to its target sites (Didon et al., 2013; Quigley and Kintner, 2016). Recently FOXN4 has been identified, which is similar to FOXJ1 needed for basal body docking at the apical membrane of multiciliated cells. In contrast to FOXJ1 loss cells can overcome defective basal body docking after FOXN4 loss. This process as well as the following steps of ciliogenesis are delayed after knock-down of FOXN4 (Campbell et al., 2016).

1.3 Model systems for studying biogenesis and function of motile cilia

Dysfunction of cilia motility leads to a group of diseases and malformations, which is collectively called PCD. In humans, this includes situs defects assitus inversus or situs ambiguous, improper clearance of the airways, male infertility and rarely hydrocephalus or female infertility. In mice hydrocephalus is observed more frequently, but the other phenotypes are comparable to the human disease. Thus, to understand molecular defects underlying PCD, regulation, biogenesis and function of motile cilia are object of intense investigations.

1.3.1 Mouse models to study consequences of motile cilia malfunction

Of the model organisms used for investigation of cilia biogenesis and function (e.g. Clamy- domonas, Paramecium,Xenopus, zebrafish), the mouse is most similar to human with respect to the tissues containing motile cilia and the pathological changes appearing due to ciliary malfunction. Thus, mice are well suited to study physiological consequences of mutations affecting genes needed for proper motile cilia function. For a better understanding of phe-

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1.3 Model systems for studying biogenesis and function of motile cilia

notypes, which might occur in cases of defective motile cilia, the function of motile cilia in mouse development will be briefly explained.

The first organ containing motile cilia in mouse development is the node, the murine left- right organizer. The node is a transient structure present approximately from embryonic day (E) 7.75 to E8.5 (Downs and Davies, 1993) and contains two kinds of cilia. Cilia of pit cells, which are located in the center of the node, display a 9+0 axoneme structure and rotational motility. They are tilted to the posterior end of the cell and generate a left-warded fluid flow by clockwise rotation (Nonaka et al., 2005; Okada et al., 2005). This flow is sensed by non-motile cilia of the crown cells, which are located at the edge of the node, and is needed to establish left-right asymmetry (Yoshiba et al., 2012). It could be shown that this system is rather robust, depending only on a few motile cilia to establish lateral asymmetry (Shinohara et al., 2012).

Another organ system depending on motile cilia for proper function are the airways. Some epithelial cells of the airways carry hundreds of cilia, which are needed to clean the airways from particles and bacteria. All epithelial cells in the nasal cavity and nasopharynx are ciliated, whereas only 20% to 40% of the tracheal epithel cells carry multiple cilia and more distally epithelia of the lung carry even fewer ciliated cells. Formation of multiple motile cilia starts around E15.5 and ciliation is complete until E18.5. Postnataly insufficient clearance of the airways leads to mucus accumulation and finally to chronic infection of airways.

In the mouse brain two types of cells carrying motile cilia are evident. Firstly, the choroid plexus forms multiciliated cells, which contain a 9+0 axoneme and show only perinatal motility. These cilia are needed to sense the amount and thus regulate the production of cerebrospinal fluid. Motility of those cilia seems not to be connected with flow of the cerebrospinal fluid (Narita et al., 2010; Nonami et al., 2013). The second type of motile cilia in the brain is formed at the apical side of the ependymal cells lining the brain ventricles and extending into the spinal canal. The ventricular system of the brain consists of two lateral ventricles, which are connected to the third ventricle, the cerebral aqueduct and the fourth ventricle, which ends in the beginning spinal central canal. Biogenesis of motile cilia starts after birth and maturation goes on until postnatal day (P) 7 (Banizs et al., 2005). Circulation of cerebrospinal fluid starts before maturation of cilia and is needed to establish complete polarization of cilia via planar cell polarity. Motility of ependymal cilia in turn keeps up the cerebrospinal fluid flow. Dysfunction of motile cilia leads to interruption of fluid flow and closure of the aqueduct, which connects the third and the fourth ventricle. Due to this blockade cerebrospinal fluid cannot be transported to the central canal, where it is usually absorbed into the lymphatic system (Ibanez-Tallon et al., 2004), leading to hydrocephalus formation.

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Both female and male fertility depend on motile cilia, which contain all a 9+2 axoneme.

Multiciliated cells in the oviducts mature around P12 and contribute to the transport of the egg towards the uterus (Dirksen, 1974; Halbert et al., 1976). In the male reproductive tract cells of the efferent duct contain multiple motile cilia, which move sperm from the testis to the epididymis (reviewed in Ilio and Hess, 1994). During this process fluid is absorbed leading to concentration of the sperm. This fluid absorption seems to facilitate transport of the sperm through the epididymis (Winet, 1980). Additionally passage through the efferent duct is needed for final sperm maturation (reviewed in Sullivan and Mieusset, 2016). The possibly most prominent cell of the reproductive systems carrying a motile cilium is the sperm. It contains a head, including the strongly condensed nucleus and the acrosome, a neck region (also called connecting piece) and a roughly 100 µm long cilium, which is termed the flagellum.

The basal body degenerates during development of the mouse sperm, in contrast to human, where it is highly reduced, but present (Manandhar et al., 1998, 2000). The flagellum itself is divided into three parts: the midpiece, the principal piece and the end-piece (figure 1.2 A). The midpiece and the principal piece are separated by the annulus, a septin-containing structure, which is thought to act as a diffusion barrier between these compartments of the sperm (Friend and Fawcett, 1974; Myles et al., 1984). In absence of the annulus, such as in Sept4 mutants, sperm exhibit reduced motility and a kinked sperm tail (Kissel et al., 2005).

Thus, the annulus is crucial for normal sperm function. The midpiece contains nine outer

Figure 1.2: Overview of sperm structure and biogenesis. AThe neck connects the sperm head to the flagellum, which consists of midpiece, principal piece and end-piece. In the midpiece the axoneme is surrounded by mitochondria and outer dense fibers, while the fibrous sheath reinforces the principal piece (from Vadnais et al., 2014)BIn the seminiferous tubules sperm develop from spermatids via mitotic and meiotic divisions and subsequent morphological differentiation (from Darszon et al., 2011).

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1.3 Model systems for studying biogenesis and function of motile cilia

dense fibers arranged around the axoneme, seven of which continue into the principal piece.

The outer dense fibers of the midpiece are surrounded by mitochondria. In the principal piece the fibrous sheath provides anchoring points for glycolytic enzymes and allows signal transduction (reviewed in Eddy et al., 2003). Outer dense fibers three and eight are replaced by the longitudinal columns of the fibrous sheath, while the rest of the outer dense fibers is continuous in the principal piece. Additionally the longitudinal columns are interconnected by transverse ribs. Outer dense fibers and the fibrous sheath appear to provide stability to the flagellum and thus constrain flagellar motion (Fawcett, 1975). Dysplasia of the fibrous sheath (DFS) is a well-described reason for male infertility (Chemes et al., 1987; Moretti et al., 2016), which was recently also termed multiple morphological abnormalities of the sperm flagella (MMAF, reviewed in Ray et al., 2016). Formation of sperm takes place embedded in the testicular Sertoli cells of the seminiferous tubules. Spermatogenesis takes approximately 35 days in mice (figure 1.2 B). During spermatogenesis sperm precursors named spermatogonia develop into mature spermatozoa. The diploid spermatogonia undergo mitosis, leading to self-renewal and formation of spermatocytes. The latter undergo two meiotic divisions and thereby form haploid spermatids. Subsequently spermatids undergo a series of morphological changes during differentiation into spermatozoa. Briefly, the acrosome is formed, the nucleus becomes condensed and reshaped, the flagellum is assembled, mitochondria are relocated to the midpiece, and excess cytoplasm is left behind in form the residual body.

In elongating spermatids the manchette, a transient microtubular structure during spermio- genesis, is needed for correct head shaping and tail formation. Subsequently the elongated spermatids line the lumen of the seminiferous tubules and are released for transport into the epididymis. Additionally two sperm maturation events are necessary before fertilization is possible. These are firstly epididymal maturation and secondly capacitation in the female reproductive tract (Austin, 1951; Chang, 1951; Young, 1931). During epididymal maturation sperm achieve motility, and the cytoplasmic droplet, which contains excess cytoplasmic material that will be removed, moves towards the annulus. Additionally protein, lipid and sugar components of the cells are modified and their composition is changed (reviewed in Gervasi and Visconti, 2017).

1.3.2 Primary cell culture of murine tracheal epithel cells as a model for multiple motile ciliogenesis

Mice are a good model system to estimate the effects of potential PCD-causing mutations.

However, biogenesis of motile cilia cannot be continuously studied in vivo, because mice need to be dissected to access ciliated cells and tissues. Hence, a method has been developed, which utilizes primary cell cultures of murine tracheal epithelial cells (mTECs) to observe

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multiple ciliogenesis in detail (You et al., 2002). Briefly, tracheal epithelial cells are isolated and selected for basal cells, which are progenitors capable of differentiation into mature airway epithelium. Various aspects of multiple ciliogenesis have been studied using this system, such as transcriptional regulation, basal body biogenesis, composition and docking as well as regeneration of injured airway epithelia (Gomperts et al., 2004; Pan et al., 2007;

Que et al., 2009; Tadokoro et al., 2014; Vladar and Stearns, 2007; Zhao et al., 2013).

1.3.3 Zebrafish as a model to study cilia function

Another popular vertebrate model to study ciliary functions is Danio rerio (zebrafish). Ze- brafish clutches can reach 50 to 200 eggs and embryos are developingex uteroin a few days.

Thus analysis of many individuals can be achieved easier compared to mice as model system.

Additionally larvae are transparent allowing detailed examination of the living embryos.

Analyses of genes contributing to ciliary function have been performed using two methods.

Classically gene expression is downregulated by injection of morpholinos, which are specifically targeted the the mRNA of interest and have initially been developed for modification ofXenopusembryos (Heasman et al., 2000). If bound to splice sites morpholinos can prevent correct splicing and thereby lead to nonsense-mediated decay. Alternatively, morpholinos can be directed to the start-codon, where they block translation. Besides morpholino-based knock-down zebrafish mutants have been generated using genetical knock-outs. Although discrepancies between knock-down and knock-out models have been observed caused both by artifacts from knock-down or knock out (reviewed by Blum et al., 2015), cilia-related genes (e.g. IFT57, IFT88) have been studied with both approaches leading to the same results (Kramer-Zucker et al., 2005; Krock and Perkins, 2008; Lunt et al., 2009). In addition to targeting of single zebrafish genes by morpholinos or genetic knock-out, screens have been performed to identify so far unidentified proteins needed for motile cilia function, such as the microarray analysis of gene expression subsequent to overexpressiom of FOXJ1 (Choksi et al., 2014a).

Zebrafish share some organs containing motile cilia with mice, namely the brain ventricles, spinal central canal and the left-right organizer, called Kupffer’s vesicle in zebrafish. Dys- function of ciliary movement in these organs leads to hydrocephalus and laterality defects as in mice (Kramer-Zucker et al., 2005). Laterality defects can easily be seen in the developing zebrafish, because the looping heart can readily be determined by visual inspection without dissection or staining of the embryos around 48 hours post fertilization (hpf). The normally rightwarded heart looping is randomized in cases of left-right defects (Chen et al., 1997;

Kramer-Zucker et al., 2005). Multiciliated cells in the spinal central canal are continuous through the whole body length in contrast to mice, but similar to humans (Alfaro-Cervello

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1.4 Identification of Cfap43 and current state

et al., 2014). Impaired cilia motility in the spinal central canal results in a curved body axis (Baye et al., 2011; Grimes et al., 2016; Kramer-Zucker et al., 2005). Scoliosis, the deformation of the spine, was also found in some cases of PCD (Engesaeth et al., 1993; Evander et al., 1983;

Juncos et al., 2014). In contrast to mice, the zebrafish carries motile cilia in the otic vesicle and in the pronephric duct. Motile cilia in the otic vesicle are needed for correct generation of otoliths, which are mineral crystal structures in the otic vesicles that serve for hearing and to sense gravity, balance and movement. If ciliary movement in the otic vesicles is disturbed, otoliths are fused, split or misspositioned within the vesicles (Colantonio et al., 2009; Wu et al., 2011). Motile cilia in the pronephric ducts are needed to move the urine from the pronephros to the cloaca. In cases of disturbed cilia function pronephric cysts can develop as well as pericardial edema due to the inability to sustain the water balance (reviewed in Swanhart et al., 2013).

1.4 Identification of Cfap43and current state

Cfap43was identified in a set of three microarrays performed by Michaela Mai (2012). In the first microarray RNA prepared from lungs from E16.5 homozygous Foxj1 knock-out mice was compared to lung RNA from heterozygous embryos, which are reported to be pheno- typically normal (Brody et al., 2000). All genes, which were downregulated, were taken into consideration. In the second microarray genes were identified, which were upregulated comparing RNA from lungs of E14.5 embryos, whose lungs contain not yet multiciliated cells, with fully ciliated lungs from E18.5 embryos. For a third microarray Notch signaling was pharmaclogically inhibited. These embryos were expected to develop more multiciliated cells in the lung by embryonic day 18.5, therefore genes that were upregulated after DAPT- treatment were identified. The intersection of these groups resulted in a candidate list of 18 genes, which were examined for their expression pattern by section in situhybridization.

Cfap43 showed strong expression in the epithelia of the conducting airways as well as in the spinal chord, the node and the adult testis. Immunofluorescence staining using a polyclonal antibody revealed potential localization of CFAP43 to sites of actin remodeling, such as the cleavage furrow and the above the apical actin web of multiciliated cells. Therefore the hypothesis emerged, that CFAP43 might act during actin-dependent processes.

In a recent study investigating male infertility several missense and nonsense mutations of Cfap43 were identified to cause DFS in sterile patients. Electron microscopy pictures of patient andCfap43knock-out mouse sperm show loss of the central microtubule pair as well as disorganization of the axonemal ultrastructure. Other phenotypes or defects of motile cilia were neither described in patients nor in knock-out mice (Tang et al., 2017).

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Table 1.1:Similarities and identities of CFAP43 protein sequences from model organisms Identity Scores (%)

H.sapiens M.musculus X.tropicalis D.rerio C.reinhardtii

Similarity Scores(%)

H.sapens 100 70.6 44.1 36.7 13.9

M.musculus 82.9 100 44.1 37.8 14

X.tropicalis 63.2 64.4 100 45 14.9

D.rerio 56.2 58.4 66.2 100 15.2

C.reinhardtii 30.3 31.2 31.7 31.5 100

Multiple alignment was performed in MacVector using the following parameters:

Matrix: Gonnet, Open gap penalty: 10, Extend gap penalty: 0.2, Delay divergent: 40%

CFAP43 is conserved in organisms from algae to human, as determined by protein sequence comparisons (see table 1.1). Therefore CFAP43 localization ofCfap43expression was additionally investigated in collaboration with Tim Ott (Institute for zoology, university of Hohenheim) using whole mount in situ hybridization in larvae of Xenopus laevis(figure 1.3 A). As in mouse, prominent expression was visible in tissues containing motile cilia, as gastrocoel roof plate, floor plate, otic vesicle and the nephrostomes of the proximal tubule as well as in the multiciliated cells of the epidermis. InXenopusthe gastrocoel roof plate is the left-right organizer, which is comparable in structure and function to the zebrafish Kupffer’s vesicle and the murine node. Cfap43 expression in the floor plate is consistent with the expression of Foxj1 and existence of motile cilia in this region (Hagenlocher et al., 2013). In contrast to zebrafish, multiciliated cells are restricted to the nephrostomes in theXenopusre-

Figure 1.3: Localisation ofCfap43inXenopus laevis(Courtesy of Tim Ott, University of Hohenheim).

AWhole mountin situhybridisation ofX. laevislarvae showedCfap43localization in tissues containing motile cilia.BGFP-tagged murine CFAP43 might localize to the apical surface of the multiciliated cells of the epidermis inX. laevis.

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1.5 Aims of this study

nal system (Tran et al., 2007). To the best of my knowledge the function of motile cilia in the floor plate, the nephrostomes and the otic vesicle is not yet described in detail. Multiciliated cells of the Xenopusepidermis are able to mediate directed fluid flow along the frog larvae.

They are similar to multiciliated cells of the airways in mammals in biogenesis and structure of the cilia, but can be investigated in the living organism, turning Xenopus into a popular model to investigate multiple ciliogenesis (reviewed in Vincensini et al., 2011). Murine GFP-tagged Cfap43 mRNA was injected into the fertilized egg to determine the subcellular localization of CFAP43. In the multiciliated cells of the epidermis CFAP43-GFP localized to the apical surface of the cells (figure 1.3 B, personal communication with Tim Ott). Thus, similar to mouse, CFAP43 expression correlates with the presence of motile cilia.

1.5 Aims of this study

The main aim of this thesis is to investigate the role of CFAP43 in biogenesis and function of motile cilia. Therefore a knock-out mouse model should be generated and analyzed to identify organs, in which the function of CFAP43 is essential. During the time needed for generation of a knock-out mouse, in vivo functions of CFAP43 should be analyzed using Danio rerio as a model system. Moreover properties of CFAP43 should be analyzed in depth in cell culture. Effects of overexpression should be analyzed in a cell culture system, which is capable of inducible overexpression of CFAP43. The same system shall be used to identify siRNAs and shRNAs, which can be used to knock-down CFAP43. Subsequently effects of knock-down could be studied in permanent and primary cell culture. For primary cell culture murine tracheal epithel cells were chosen, because CFAP43 was reported to be expressed in those cells (Mai, 2012). Therefore, this cell culture system, which was developed by You and Brody (2012), needed to be established. Additionally new antibodies should be generated, either to confirm the results from the previously generated antibody or in case of contradictions, to identify an antibody which detects endogenous CFAP43 in immunofluorescence staining on cultured cells or tissue sections. To identify pathways or protein complexes in which CFAP43 might act, more potential interaction partners should be identified using tandem-affinity purification and subsequent identification of co-purified proteins via mass spectrometry. Subsequently newly identified potential binding partners as well as proteins previously identified in the yeast-two-hybrid screens should be validated by co-immunoprecipitation.

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2.1 Analysis of CFAP43 in Danio rerio

Zebrafish is a model system often used to investigate the function of proteins using knock- down studies. Benefits are the ex utero and fast development, as well as the transparent embryos. Therefore changes of the morphology are easily visible during embryonic development. In addition morpholino-based knock-down can be achieved by direct injection into the fertilized egg and thus allows a fast system to manipulate protein levelsin vivo. Phenotypes connected to cilia defects are well described and technically easy to study. As mentioned before,Cfap43 is not only existent in mice, but conserved between diverse animals. Hence, Danio reriowas chosen as a model system to initially investigate effects ofCfap43knock-down.

2.1.1 Expression analysis ofCfap43in zebrafish embryos

First, expression of Cfap43 in the early embryo was investigated using RT-PCR and in situ hybridization. Cfap43 was detected in all cDNA preparations from 4 to 32 hpf (figure 2.1 A), thus it was expressed in non-differentiated cells of the early embryo and distribution of mRNA might be restricted to distinct tissues during development. To investigate the localization of the mRNA in 24 hpf embryos, whole mountin situhybridization was performed with various probes directed againstCfap43. BecauseMyl7expression was described to be clearly restricted to the heart, a probe detectingMyl7was used as positive control for the experimen- tal setup (figure 2.1 C(f); Yelon et al., 1999, personal communication with Dr. Timm Haack).

Detection with each of the probes resulted in staining of the whole embryos, partly with a stronger signal in the head (figure 2.1 C(a-e)). To specifically verify expression of Cfap43in a tissue containing motile cilia, pronephric ducts were isolated as described by Drummond and Davidson (2009). RT-PCRs for forCdh17, which is restricted to the pronephric duct and FoxJ1a, which is expressed in tissues containing motile cilia, such as the pronephric duct, spinal central canal, otic vesicle, brain ventricle, or Kupffer’s vesicle, were established using cDNA preparations of whole embryos. Cdh17 and Foxj1a transcripts were detected in the isolated tissue, proving successful preparation of pronephric ducts. Finally, expression of Cfap43 was shown by RT-PCR on the isolated tissue (figure 2.1 B, asterisk). Thus, Cfap43 expression in the pronephric ducts could be proven.

2.1.2 Knock-down studies in zebrafish

To study the function of CFAP43 in zebrafish, various morpholino oligonucleotides directed againstCfap43were designed. First, two morpholinos (i1 morpholino, MO i1; i4 morpholino,

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2.1 Analysis of CFAP43 in Danio rerio

Figure 2.1: Expression ofCfap43inDanio rerio. ACfap43was detected in zebrafish embryos from 4 to 32 hpf.BCdh17andFoxJ1awere detected in whole embryos as well as in isolated pronephric ducts.

Cdh17expression was restricted to pronephric ducts, thus isolation of pronephric ducts was successful (Thisse et al., 2001). In addition Cfap43 can be detected in isolated pronephric ducts (asterisk).

CWhole mountin situhybridization with five distinct probes (a-e) resulted each in overall staining of the embryos, partly with a stronger signal in the head. A probe againstMyl7 (f, red arrowhead), which localizes to the heart, was used as positive control.

MO i4), which bind the splice acceptor sites between intron 1 and exon 2 or intron 4 and exon 5, and should prevent splicing from exons 1 to 2 or exons 4 to 5. The resulting transcripts contain premature stop-codons and should therefore result in nonsense-mediated decay of the mRNA (for review see Nicholson and Mühlemann, 2010). Effective splice inhibition as well as RNA degradation was tracked by RT-PCR. Figure 2.2 A shows the Cfap43 mRNA containing 38 exons and primer pairs, which were used to control the outcome of splice morpholino (MO i1 and MO i4) injection. The first two primer pairs (#256+#257 and #258+#259) were used to investigate the direct effect of the morpholino i1 on the transcript. The PCR spanning intron 1 resulted in an upward shift of the PCR product subsequent to injection of MO i1, as expected if the intron is included in the product. In contrast, the PCR spanning exons 2 to 4 showed a downward shift after injection. Cloning and sequencing of this new and unexpected product revealed a transcript, which was shortened by 90 base pairs. This deletion led to a protein, which is shortened by 30 amino acids (1612 instead of 1642 amino acids) without any premature stop codons (figure 2.2 B, asterisk). In addition quantitative RT-PCR revealed no knock-down of CFAP43 in fish injected with morpholino i1 compared to non-injected individuals, and no morphological abnormalities were observed subsequent

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Figure 2.2: Knock-down and ectopic expression ofCfap43inDanio rerio. AThe effect of splice site morpholino injection was determined by RT-PCR using primer pairs #256+#257, #258+#259 (MO i1) and #276+#277 (MO i4). Primers #266+#267 were designed for quantitative RT-PCR.BRT-PCR from exon 1 to 2 resulted in a band of 247 base pairs in control embryos and an additional band of 520 base pairs in the injected individuals, demonstrating splice inhibition. RT-PCR spanning exons 2-4 resulted in a shortened product (asterisks) in the injected fish compared to the control, indicating a possible alternative splice event. CRT-PCR spanning exons 4-6 reveals bands of 293 bp (exons 4, 5 and 6) and 418 bp (exons 4, 5, 6 and intron 4), matching the expected PCR products. Thus the injected morpholino prevented splicing from exon 4 to 5. D Quantitative RT-PCRs of eight independent injections revealed knock-down of complete Cfap43 mRNA to about 60% compared to uninjected embryos.EMurineCfap43could be detected at least until 32 hpf after injection of 60 ng mRNA.

to injection with MO i1 in developing zebrafish embryos until 96 hpf. Thus injection with the i1 morpholino did not disrupt functional CFAP43. In contrast, after injection of MO i4 the PCR spanning exons 4-6 (primers #276+#277) revealed only the upward shift expected when including intron 4, but no unexpected additional bands (see figure 2.2 C). The higher band resulting from splice inhibition became more prominent with increasing amounts of injected morpholino. Since injection of high amounts of i4 morpholino led to death of the embryos by toxic side effects of the morpholino, only 5 ng of this morpholino were injected in the knock-down experiments. After each injection quantitative RT-PCR was used to mon- itor the knock-down effect. The average knock-down effect determined in nine independent

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experiments was approximately 60% (figure 2.2 E; table 5.1, appendix).

Figure 2.3 (column "MO i4") shows the result of Cfap43 knock-down by the splice site morpholino. An overview over a batch of fish (A) shows the usual straight body axis of non-injected individuals (a, f), which is indistinguishable from the appearance of i4 morpholino injected fish (b, g). Strong body axis curvature as observed after knock-down of other cilia-related genes (e.g. Kramer-Zucker et al., 2005; Ryan et al., 2013) was not achieved in this situation. Pericardial edemas were found in 14% of the individuals (figure 2.3 B (g), representative picture of one individual, arrowhead). Pericardial edemas were described to develop due to defective water homeostasis in cases of disturbed pronephros or pronephric duct function in the absence of cilia motility (reviewed in Swanhart et al., 2013). In addition to pericardial edema few individuals from knock-down experiments displayed hydrocephali (4%), which are visible in magnifications of the heads (figure 2.3 C (l)). Miss-arranged otoliths were observed after injection of the i4 morpholino in 11% of the individuals (figure 2.3 D (u)), which can be explained by the failure of proper otolith positioning by motile cilia. For analysis of the pronephric ducts zebrafish expressing murine ARL13B-GFP were used, which allowed imaging of the cilia without the need of additional staining procedures. For better visualization of the pronephric ducts fish were stained with fluorescently labeled phalloidin, which binds to F-actin and thus marks among others the apical surface of polarized epithel cells. Confocal Z-stacks were taken for evaluation of the pronephric ducts and their cilia and overlays generated using ImageJ. Ducts of the knock-down fish appeared slightly dilated and cilia orientation, which was very uniform in the non-injected controls, seemed to be randomized (figure 2.3 E). To quantify the effect of knock-down, the pronephric duct width was measured at several points along the duct and averaged for each individual. The results are shown as a dot for each fish in a scatter plot (figure 2.3 G; for raw data see table 5.3, appendix).

To validate the knock-down results described above, another morpholino directed against the start-codon was used to block translation (ATG morpholino, MO ATG), without affecting transcript structure. Antibodies detecting CFAP43 in zebrafish were not available, therefore this knock-down could not be proven. However, ATG morphants recapitulated the phenotypes observed in i4 morphants, suggesting that MO ATG effectively blocked translation.

In addition to the phenotypes observed in i4 morpholino injected animals, many ATG morpholino injected fish (84%) displayed a curved body axis (figure 2.3 A (d), B (i) and F (dark blue column)). As for i4 morpholino injected individuals, pericardial edema (78%) and hydrocephali (25%) were observed after injection of the ATG morpholino (B (i) and C (n)), but occurred with a higher frequency. Magnifications of the otic vesicles reveal malformations as fused or surplus otoliths (66%, figure 2.3 D (y-a’)). As already observed for body axis curva-

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ture, otoliths defects appeared to be more severe and occurred more often in animals, which were injected with the ATG morpholino, as compared to the i4 morpholino. Also dilation of the pronephric ducts and miss-orientation of its cilia was more prominent in comparison to the splice morpholino injected animals (figure 2.3 E). In addition to the phenotypes depicted in figure 2.3, laterality was investigated in 48 hpf embryos by evaluation of the direction of heart looping in clutches of three independent injections. No randomization of heart looping could be monitored, neither in fish injected by i4 morpholino nor by ATG morpholino.

Thus, in zebrafish CFAP43 seems not to be necessary for determination of the left-right axis.

Although cilia of the Kupffer’s vesicle seem to perform their function normally, all the other phenotypes observed after injection of the morpholinos have been linked to defects of ciliogenesis or cilia motility before (Grimes et al., 2016; Kramer-Zucker et al., 2005; Swanhart et al., 2013; Wu et al., 2011).

To control the specificity of the knock-down effects, rescue experiments were performed by co-injection of murine Cfap43 mRNA with each of the morpholinos. The murine transcript was chosen, since the morpholinos were specific for the sequence of zebrafishCfap43.

Therefore the morpholinos could neither promote a knock-down of the murine mRNA nor could the RNA promote a rescue effect by binding and titration of the morpholinos. Figure 2.2 D shows the presence of the murine mRNA until 32 hpf, thus a potential rescue effect should be visible at least until this stage of development. Body axis curvature of fish, which were injected with the i4 morpholino and murine mRNA, was comparable to the results seen after injection of the i4 morpholino alone (figure 2.2 A (b, c) and F). In contrast, the curved body axis seen in ATG morpholino injected embryos appeared to be milder in the rescue situation (ATG morpholino +Cfap43 mRNA) and fewer individuals were affected (84% vs.

51%, figure 2.3 A (d, e), B (i, j) and F). Pericardial edema (MO i4: 14% vs. 6%, MO ATG: 78%

vs. 51%), hydrocephali (MO i4: 4% vs. 1%, MO ATG: 25% vs. 8%) and otolith defects (MO i4:

11% vs. 3%, MO ATG: 66% vs. 27%) were observed with lower frequencies after co-injection of the Cfap43 RNA compared to the knock-down situations described above. Furthermore dilation of the pronephric ducts could be partially rescued by co-injection of the mRNA with the morpholinos. After rescue of the i4 morpholino the pronephric ducts had an appearance similar to wild type ducts, and also treatment with the ATG morpholino together with the RNA led to slimmer ducts than injection of the ATG morpholino alone. The frequencies of the observed phenotypes after morpholino-mediated knock-down and the corresponding rescue experiments are summarized in the graph in figure 2.3 F and the averages of the measured pronephric duct widths are shown in 2.3 G. The corresponding numbers are shown in table 5.2 in the appendix. Body axis curvature, pericardial edema and hydrocephali were evaluated in five independent experiments using between 20 and 100 individuals for each

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Figure 2.3: Effects ofCfap43knock-down in Danio rerio. AOverview images of 100 hpf zebrafish embryos show straight body axes in non-injected as well as most of the splice-morpholino (+ murine Cfap43mRNA) injected embryos. In contrast, many of ATG-morpholino injected embryos displayed a curved body axis, which appeared to be milder after injection of Cfap43 mRNA. B Pictures of representative embryos allow show the body axis and reveal pericardial edema (arrowhead).CMag- nifications of the heads show hydrocephali seen in some i4- and ATG-morpholino injected animals. D Examples of otoliths are shown for differently treated zebrafish embryos. Untreated embryos formed two otoliths in each otic vesicle, in the knock-down situation fused, split or miss-arranged otoliths could be observed (arrowheads). EConfocal images of the pronephric duct of 32 hpf embryos show a slim tube with uniformly oriented cilia in non-injected fish. Pronephric ducts of splice- or ATG- morpholino injected animals were dilated and cilia seemed to be more randomly oriented. These phenotypes appeared milder after co-injection ofCfap43mRNA.F Quantitative analysis of observed phenotypes is depicted in a bar diagram. GWidth of pronephric duct was determined by measuring the distance between phalloidin-marked cell surfaces at several points in each confocal image. The resulting plot indicates that knock-down ofCfap43led to dilated pronephric ducts, which could partly be rescued by co-injection with murineCfap43mRNA.

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condition. Otolith formation was investigated in three experiments and pronephric ducts of 16 to 23 individuals were imaged in three (controls, morpholino knock-down) to four (rescue) independent experiments.

In addition to the above mentioned tests, the length of cilia in the spinal central canal and pronephric duct were measured using confocal images of both organs. In this experiment non-injected fish were compared to animals treated with i1 morpholino or ATG morpholino.

Both types of injected fish displayed cilia shortened about 10%. During analysis of those experiments it became apparent, that i1 morpholino did not lead to knock-down ofCfap43(see figure 2.2 B), thus shortened cilia were an artifact produced by injection of the morpholinos, rather than a true phenotype ofCfap43knock-down.

Figure 2.4: Effect of CFAP43 on cilia length. A Average length of ponephric duct cilia of 24 hpf zebrafish embryos was reduced after morpholino injection. BAverage length of spinal central canal cilia of 72 hpf zebrafish embryos was reduced after morpholino injection.

In summary, knock-down of CFAP43 could be achieved using two different morpholinos, whereas the third morpholino led to alternative splicing products that can generate functional protein. CFAP43 knock-down resulted in developmental defects that have been associated with defects in cilia motility before and could be partially rescued by expression of murine CFAP43. Thus, using the zebrafish as a model system not only evidence for CFAP43 function in motile cilia could be shown, but also the functional conservation between CFAP43 proteins from fish and mouse.

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2.2 Generation and characterization of antibodies against CFAP43

2.2 Generation and characterization of antibodies against CFAP43

For analysis of a protein antibodies are essential tools to detect expression of endogenous protein in various tissues and cell lines, to precipitate the protein for identification of interaction partners and to determine the subcellular localization in immunohisto- and immuno- cytochemical staining. Therefore a set of antibodies directed against different peptides of CFAP43 was generated and characterized.

2.2.1 Evaluation of polyclonal antibodies

Figure 2.5 A shows an overview about the general architecture of CFAP43 based on structure prediction, including seven WD-repeats in the N-terminus and three coiled-coil domains in the C-terminus (Mai, 2012). Polyclonal antibodies against six different peptides, which are distributed along the whole protein sequence, were generated by Michaela Mai. Antibodies against peptides 1 to 4 have been extensively studied by Michaela Mai (2012). The antibodies against peptide 1 (P1) detected overexpressed CFAP43 in western blots. Antibodies against peptide 4 (P4) detected overexpressed protein in western blots and immunofluorescence and, potentially, endogenous CFAP43 in immunohistochemistry and immunocytochemistry.

Additionally antibodies directed against peptides 5 and 6 (P5.1, P5.2 and P6) were evaluated in the context of this thesis. Their ability to detect CFAP43 was evaluated in western blots containing immunoprecipitated CFAP43 that was overexpressed in CHO cells and a crude lysate of non-modified CHO-cells (figure 2.5 B). The western blot was divided into four parts, one was probed with P4 as positive control, the others were probed with P5.1, P5.2 and P6. The antibody P4 detected the expected protein of approximately 186 kDa in the immunoprecipitation from CHO cells overexpressing CFAP43, but not in the lysate of untransfected cells. In contrast, none of the other antibodies detected immunoprecipitated CFAP43. Instead, P5.1 and P5.2 detected a protein in CHO cell lysates with an apparent molecular size similar to CFAP43. This protein does not represent CFAP43 because it was not detected in the precipitate.

To further characterize the P4 antibody, a murine cell line was used (described in detail in sections 2.4.1.1 and 2.4.1.3), in which CFAP43 overexpression can be induced by treatment with doxycyclin. In addition knock-down was achieved after transduction of siRNAs directed against Cfap43. These tools were used to further evaluate the antibody P4, which showed the most promising results so far. In figure 2.6 A overexpression is clearly visible comparing doxycyclin-induced vs. untreated cells (lanes 7 and 8), whereas knock-down is mediated by different siRNAs, with a combination of siRNA 2 and 3 having the strongest effect (compare lanes 1 to 6). With these cells I compared the wild type (showing only en-

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Figure 2.5: Characterization of polyclonal antibodies against peptides. ALocalization of peptides used for production of polyclonal antibodies. B Antibodies directed against peptides 5 and 6 (P5.1, P5.2 and P6) were probed against a western blot of immunoprecipitated CFAP43 (transiently overexpressed in CHO cells, expected size marked by green box) and non-transfected CHO cells. None of them detected precipitated CFAP43. As a control an antibody against peptide 4 (P4) was used, which is known to detect CFAP43 in western blot.

dogenous protein), knock-down and overexpression situation in immunofluorescence (figure 2.6 B). In murine cells expressing only endogenous CFAP43 protrusions could be shown, as described by Mai (2012) (marked by asterisks). Those protrusions were not vanishing in the knock-down situation (a vs. b), although the siRNAs were shown to reduce the amount of overexpressed CFAP43 in this cell line (see section 2.4.1.3). Therefore this protrusions were most likely staining artifacts not connected to CFAP43. In immunofluorescence doxycyclin- mediated overexpression could clearly be shown (d). However, the fluorescent signal detected in overexpressing cells, which were treated with siRNAs2+3 (e), appeared only slightly reduced compared to the signal from control cells (d and f). In contrast, in western blot signal intensities from cells treated in the same way was clearly different (figure 2.6 A). Thus, the antibody P4 clearly detects overexpressed CFAP43, but might produce artifacts when used for detection of the endogenous protein in immunofluorescence.

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2.2 Generation and characterization of antibodies against CFAP43

Figure 2.6: Knock-down of CFAP43 in cultivated cells. A Western blot analysis of cells with and without induced CFAP43 expression as well as transfected with various siRNAs revealed strong differences between the signals of endogenous CFAP43, doxycyclin-induced overexpression and knock- down of the overexpressed protein. BIn immunocytochemistry these differences could only partly be reproduced. The "protrusions" described by Mai (2012), marked by asterisks, do not disappear by siRNA-mediated knock-down.

2.2.2 Generation and evaluation of monoclonal antibodies

None of the polyclonal antibodies detected endogenous CFAP43 convincingly in immunocytochemistry so far. Therefore monoclonal antibodies were generated against a purified fragment of CFAP43 expressed in bacteria. Three expression constructs (see figure 2.7 A) were prepared comprising fragments of CFAP43, which would based on structure predictions be expected to fold correctly independently from the rest of the protein. These fragments were MBP-tagged, because MBP was reported to increase the solubility of proteins (Kapust and Waugh, 1999) and earlier attempts of CFAP43 overexpression and purification resulted in insoluble protein (Mai, 2012). Expression of the coiled-coil domain (referred to as fragment 2, figure 2.7 A) resulted in the highest protein amount, but non of the fusion constructs was soluble under the tested conditions. Thus, monoclonal antibodies were generated using fragment 2 for immunization. The fusion protein was expressed in E. coliunder control of an IPTG-inducible promoter. The fractions resulting from protein overexpression and purification are shown in figure 2.7 B. When comparing the lysates of non-induced vs. induced

Characterization of CFAP43 and its function in motile cilia

Medizinische Hochschule Hannover leerzeile

Institut für Molekularbiologie

Characterization of CFAP43 and its function in motile cilia

INAUGURALDISSERTATION

zur Erlangung des Grades einer Doktorin der Naturwissenschaften - Doctor rerum naturalium -

(Dr. rer. nat.)

Contents

Zusammenfassung

Abstract

1 Introduction