VERA SHIROKOVA Foxi3 in Hair Follicle Development and Homeostasis Recent Publications in this Series

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Helsinki 2016 ISSN 2342-3161 ISBN 978-951-51-2726-6 Transcription Factor Foxi3 in Hair Follicle Development and Homeostasis

Vera Shirokova

Division of Genetics Department of Biosciences Faculty of Biological and Environmental Sciences

and

Doctoral Program in Integartive Life Science University of Helsinki

ACADEMIC DISSERTATION

To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki in auditorium 1041 in Biocenter 2, Viikinkaari 7 on December 9th 2016, at 12 o’ noon.

Helsinki 2016 Supervised by Docent Marja Mikkola University of Helsinki, Finland

Advisory committee Docent Ulla Pirvola University of Helsinki, Finland

Professor Antti Iivanainen University of Helsinki, Finland

Pre-examiners Professor Antti Iivanainen University of Helsinki, Finland

Professor Seppo Vainio University of Oulu, Finland

Th esis opponent Professor Vladimir Botckarev University of Bradford, UK Boston University School of Medicine, USA

Custodian Professor Juha Partanen University of Helsinki, Finland

Published in Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis

ISBN 978-951-51-2726-6 (paperback) ISBN 978-951-51-2727-3 (PDF) ISSN 2342-3161 (Print) ISSN 2342-317X (Online)

Cover layout by Anita Tienhaara Press: )BOTB1SJOU7BOUBB “Th e embryo is fi lled with an internal psyche and wisdom to accomplish its goals despite all the obstacles embryologists placed in its path”

Hans Dreisch 1893

Александрам TABLE OF CONTENTS

List of original publications ...... vi Abbreviations ...... vii Summary ...... ix Introduction ...... x 1 REVIEW OF THE LITERATURE ...... 1 1.1 Organs formed as ectodermal appendages ...... 1 1.2 Hair follicle as an ectodermal appendage ...... 1 1.2.1 Mouse hair types ...... 2 1.2.2 Morphological stages of hair follicle development...... 3 1.2.3 Th e hair cycle ...... 5 1.2.4 Telogen hair follicle ...... 6 1.3 Molecular regulation of hair follicle development and renewal ...... 8 1.3.1 Wnt pathway...... 8 1.3.2 Bmp pathway ...... 9 1.3.3 Activin pathway ...... 13 1.3.4 Ectodysplasin pathway ...... 14 1.3.5 Shh pathway ...... 15 1.3.6 Fgf pathway ...... 16 1.4 Key transcription factors regulating hair follicle stem cells ...... 17 1.4.1 Sox9 – pioneer factor and master gene regulator of hair follicle stem cells ...... 18 1.4.2 Lhx2 – a signpost at the matrix/sebocytes crossroads ...... 19 1.4.3 Nfatc1 – quiescence and aging of hair follicle stem cells ...... 22 1.4.4 Runx1 – an activator of anagen onset ...... 22 1.5 Coordination of hair follicle stem cell regulation ...... 24 1.6 Hair follicle stem cell specifi cation ...... 25 1.7 Mammary gland as an ectodermal appendage ...... 26 1.8 Ectodermal dysplasias and hair disorders ...... 27 1.8.1 Monogenic hair disorders in humans ...... 27 1.8.2 Hypohidrotic ectodermal dysplasia ...... 28 1.8.3 Animal models in the study of ectodermal dysplasia and genetic hair disorders ...... 30 1.9 Forkhead box transcription factors ...... 30 1.9.1 Fox transcription factors in the hair follicle ...... 31 1.9.2 Foxi3 ...... 31 2 AIMS OF THE STUDY ...... 33 3 MATERIALS AND METHODS ...... 34 4 RESULTS AND DISCUSSION ...... 36 4.1 Expression pattern of Foxi3 in ectodermal appendages (I and II) ...... 36 4.1.1 Foxi3 displays dynamic expression in developing and cycling hair follicles (I and II) ...... 36 4.1.2 Multiple pathways regulate Foxi3 expression (I and unpublished) ...... 36 4.2 Impact of Foxi3 deletion on ectodermal appendage development (II and unpublished) ...... 38 4.2.1 Hair follicle downgrowth and hair cycling were aff ected in the absence of Foxi3 (II) ...... 38 4.2.2 Foxi3 is dispensable for mammary gland development (unpublished) ... 41 4.3 and pathways downstream of Foxi3 (II and unpublished) ...... 42 4.3.1 A number of hair follicle stem cell specifi c genes are downstream of Foxi3 (II and unpublished) ...... 42 4.3.2 Foxi3 regulates hair follicle stem cell specifi cation during morphogenesis ...... 43 4.3.3 Foxi3 is required for hair follicle stem cell maintenance and activation ..45 4.4 Tsr2 is a novel regulator of hair follicle development and growth ...... 46 5 CONCLUSION AND FUTURE PERSPECTIVES ...... 48 6 ACKNOWLEDGEMENTS ...... 50 7 REFERENCES ...... 52

LIST OF ORIGINAL PUBLICATIONS

I Shirokova V*, Jussila M*, Hytönen MK*, Perälä N, Drögemüller C, Leeb T, Lohi H, Sainio K, Th esleff I, Mikkola ML. 2013. Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes. Developmental Dynamics. 2013 Jun;242(6):593-603. *equal contribution

II Shirokova V, Biggs LC, Jussila M, Ohyama O, Groves AK, Mikkola ML. 2016. Foxi3 defi ciency compromises hair follicle stem cell specifi cation and activation. Stem Cells. 2016 Jul;34(7):1896-908. doi:10.1002/stem2636

III Murgiano L, Shirokova V, Welle MM, Jagannathan V, Plattet P, Oevermann A, Pienkowska-Schelling A, Gallo D, Gentile A, Mikkola M, Drögemüller C. 2015. Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation. PLoS Genetics. 2015 Jul 23;11(7): e1005427.

Th is thesis is based on original publications, which are referred to in the text by their Roman numerals.

vi ABBREVIATIONS

APC Adenomatous polyposis coli tumor suppressor protein Bmp Bone morphogenetic protein BrdU Bromdeoxyuridine ChIP Chromatin immunoprecipitation cKO Conditional knock-out mouse DBD DNA-binding domain Dkk Dickkopf E Embryonic day ECM Extracellular matrix ED Ectodermal dysplasia Eda Ectodysplasin Edar Ectodysplasin Edaradd Edar-associated death domain FACS Fluorescence activated cell sorting Fgf Fibroblast growth factor Fox Forkhead box DNA-binding domain containing transcription factor Gli Glioma-associated oncogene homolog HED Hypohidrotic ectodermal dysplasia Hh Hedgehog protein family Id Inhibitor of DNA binding IKK IκB kinase complex ICR Mouse strain originated from the Institute of Cancer Reserach, Philadelphia ISH In situ hybridization IκB Inhibitor of kappa B K KGF Keratinocyte growth factor KO Knock-out mouse Lef-1 Lymphoid enhancer-binding factor 1 Lgr Leucine-rich repeat-containing G protein-coupled receptor Lhx Lim- containing transcription factor Lrp Low-density lipoprotein receptor-related protein Msx Msh homeobox containing transcription factor Nfatc Nuclear factor of activated T-cells NF-κB Nuclear factor kappa-B NMRI Mouse strain originated from the Naval Medical Research Institute, Maryland OE Overexpression Pax Paired box homeodomain containing transcription factor PTHrP Parathyroid hormone-related protein qRT-PCR Quantitative real time PCR R-spondin Reelin domain-containing spondin Runx Runt related transcription factor SC Stem cell Shh Sonic hedgehog

vii SNP Single nucleotide polymorphism Sostdc1 Sclerostin domain-containing 1 Sox SRY-related high-mobility group box containing transcription factor Tcf T-cell transcription factor Tgf β Transforming growth factor β TNF Tumour necrosis factor Ubx Ultrabithorax homeobox containing transcription factor XLHED X-linked hypohidrotic ectodermal dysplasia -/- or +/- Homozygote or heterozygote animal for a mutated gene

Gene names are written in italics and protein names in roman. Mouse genes or are in lower case letters and human in UPPER CASE letters.

viii Summary

Development of the organ and its regeneration in adulthood are highly related events and are oft en guided by similar molecular cues. Th e hair follicle undergoes repetitive cycles of growth and represents an ideal model for studying both development and regeneration. Hair follicle is an ectodermally-derived organ formed through interaction of embryonic epithelium and mesenchyme. Once morphogenesis is accomplished, hair follicle undergoes subsequent cycles of growth, regression and rest. Hair regeneration is ensured by the population of hair follicle stem cells which are set aside early during embryonic development. Th e most quiescent stem cells reside in the bulge, while the stem cells fated for the new growth are in the hair germ.

Transcription factors control gene expression and regulate the variety of cell fate choices. Transcription factor Foxi3 is a causative gene for hairless phenotype in several dog breeds, but little else is known about its function in the hair follicle.

In this thesis work I have studied the role of Foxi3 in hair follicle development and postnatal regeneration. To address this question several Foxi3-defi cient mouse lines were used, as well as gene expression analysis, tissue culture techniques and molecular biology methods. Foxi3 is expressed in the developing hair follicle and in the hair germ, exclusively in the activated stem cells. Foxi3 is essential for specifi cation of the hair follicle stem cells and for their activation at the beginning of the hair growth. Foxi3-defi cient mice have delayed hair follicle downgrowth during embryogenesis. In the absence of Foxi3, postnatal natural hair regeneration and hair regrowth aft er depilation are both impaired due to poor activation of stem cells and decrease in their number. Genome-wide profi ling, quantitative PCR and immunostaining showed downregulation of several stem cell associated genes upon Foxi3 loss. Th us, Foxi3 is a novel regulator of hair follicle stem cell specifi cation, maintenance and activation.

ix Introduction

Developmental biology encompasses not only the questions of embryonic development: how the fertilized egg develops into the whole body and how that adult body produces yet another body, but it also aims to understand the processes of regeneration. Th e phenomenon of regeneration can occur at the level of the whole organism, exemplifi ed by the ability of hydra and planaria to restore the whole body from a cell suspension or salamander’s capacity to regrow a full limb. Regeneration also encompasses maintaining the functionality of organs and tissues of the adult organism to replenish constant cell loss and ensure wound healing. Th ese processes are possible due to the stem cells (SCs), which reside in the adult body from embryogenesis and activate their vital potential upon need. Elucidating the mechanisms controlling SC quiescence and activation are one of the main scientifi c targets of modern developmental biology (Gilbert, 2014). Development of the organism is regulated by genetic programs, which defi ne the time when the gene will be activated or silenced. Th ese genetic programs are organized as gene regulatory networks, where a change in the activity of one gene aff ects the functional state of downstream genes, leading to activation or inhibition of the developmental process. Ultimately, these changes result in cellular behaviours such as proliferation, diff erentiation, and migration, thus directing the developmental process in a way predetermined by genetic program. DNA harbours information about all the genetic programs of the organism, the realisation of this information is a function of diff erent proteins which interact with the DNA. As science journalist N. Angier has written, “DNA is more like a certain type of a politician, surrounded by a fl ock of proteins handlers and advisors that must vigorously message it, twist it, and on occasion, reinvent it before the grand blueprint of the body can make any sense at all” (Angier, 2003). Th ese handlers and advisors are the transcription factors, which recognise specifi c DNA sequences and by that activate or repress transcription of their target genes. Transcription factors are grouped into families based on their DNA-binding domain (DBD): homeobox-containing (Ubx, Pou, Pax, etc), Forkhead box-containing (Fox proteins, including Foxi3), Rel homology region-containing (NF-κB, Nfatc, etc), High-Mobility-Group region-containing (Sox, Tcf, etc), basic helix-loop-helix transcription factors and many others (Gilbert, 2014). From the earliest stages of development through adult, cellular behaviours such as adhesion, migration, diff erentiation and proliferation are regulated by reciprocal cell signaling. Cells communicate through membrane-associated juxtacrine factors and diff usible paracrine factors. Paracrine factors work in a wider range than juxtacrine signalling, they diff use for about 15 cell diameters, 40-200 μm (Bollenbach et al., 2008; Harvey and Smith, 2009). Th e main focus of this work will be on paracrine signalling since it has been studied intensively, and currently more developmental processes are known to be controlled by paracrine than juxtacrine signalling. Th e induction and morphogenesis of organs is regulated by a relatively small set of paracrine factors. Th e embryo inherits a rather compact, evolutionarily conserved, genetic “tool kit” and uses it to construct the heart, kidney, glands, to grow hairs and teeth. Almost all paracrine factors can be grouped into four families based on their function and mechanism of action:

x 1. Th e fi broblast growth factor (Fgf) family 2. Th e Hedgehog (Hh) family 3. Th e Wnt family 4. Th e TGF-β superfamily, comprising TGF-β and activin families, bone morphogenetic proteins (Bmp), and some others.

All paracrine signalling functions according to a common scheme. Paracrine factors, also called ligands, bind to a receptor that initiate a series of enzymatic reactions within the cell. Th e end point of these reactions is the expression of certain genes. Th ese could be the genes of transcription factors which will in turn activate or repress the expression cell-specifi c genes that drive cellular diff erentiation. It could also be a direct regulation of some eff ector genes leading to changes in cellular proliferation or adhesion. All the consequence of the reaction events in the cell caused by the response to the paracrine factor is called signalling pathway. Th ese pathways link ligand binding to the cell membrane to gene expression changes in the nucleus and are the fundamental modules of development and also all other processes in the living organism (Gilbert, 2014). Later each signalling pathway downstream of four families of paracrine factors will be discussed step-by-step, with the focus on their actual function in relation to this study. Development provides a time-window for studying gene function in which the image of signaling interactions looks brighter and sharper. Th is is due to the speedy character of development, when the whole genetic programs of organ and tissue formation must unfold properly in a certain restricted time period. During development the impact of certain genes are more prominent. Th e adult tissues mainly persist in a steady state of homeostasis, since their aim is to keep a status quo without gross transcriptional changes. Regeneration resembles developmental process, however, there is an input from the surrounding tissue which aff ects cellular behaviour. In developing organism, cell and tissue positions and functional states are oft en transient, and multiple regulatory pathways need to converge in coordination. Consequences of gene ablation are usually more prominent and faster to register. Even though certain genes have diff erent roles in development and in terminally diff erentiated tissues, characterization of the gene function solely in diff erentiated tissue incomplete. Th is PhD thesis focuses on the molecular control of hair follicle development and homeostasis and also covers some aspects of mammary gland biology. Th e following chapters of the literature review will give a detailed description of the biology of the hair follicle. Th e development and postnatal modifi cations of the mammary gland will be discussed briefl y as well.

xi Review of the literature 1 REVIEW OF THE LITERATURE 1.1 Organs formed as ectodermal appendages A group of organs of the body develop as an appendages of the surface ectoderm and are therefore called ectodermal organs, or ectodermal (skin) appendages. Th ey include hair follicles, teeth, a number of glands (mammary, salivary, sweat, lacrimal), nails, scales, and feathers. Being at the surface of the body, ectodermal organs have a defi nite adaptive signifi cance and are exposed to intensive natural selection. Due to that, ectodermal organs oft en represent those evolutionarily derived systematic traits that defi ne taxonomical borders, such as hairs and mammary glands for mammals and feathers for birds. Th e evolution of each group of ectodermal appendages has been tightly controlled and resulted in a great number of diff erent ectodermal organs and in a variety within each organ type. One can think about the manifold types of mammalian hairs and bird feathers, the variety of shapes of fi sh scales. At the same time, these organs are much more similar than it could be guessed from their appearance and the functions they carry. Th e common route is in their development, which proceeds through similar principal steps and requires tightly controlled interaction between epithelium and underlying mesenchyme (Biggs, Mikkola, 2014; Hughes et al, 2011). Th e fi rst morphological sign of ectodermal appendage formation is an epithelial thickening, called a placode. Growing tooth, hair and glandular placodes deepen into the mesenchyme and form a bud. From there on, unique structures are formed in the course of morphogenesis of each ectodermal organ. Th e function of the organ in the adult organism dictates the mode of its homeostasis aft er formation, e.g. regeneration capacity, metabolic characteristics, and periods of activation and dormancy (Geyfman et al, 2014). Skin appendages are ideal models to help answer many fundamental biological questions. Skin appendages are excellent experimental models because they are at the body surface, or close to. Many of these appendages are accessible to experimentation, and easy to observe. Th ey develop relatively late in embryogenesis, and many of them undergo physiological regeneration in adult life. Since there are many appendages on one organism, alterations are unlikely to be lethal, allowing more opportunities for perturbation (Hughes et al., 2011). However, while more and more of appendage biology becomes defi ned, we still cannot make an epidermal appendage in a culture dish, nor in engraft ed skin (Fuchs, 2016). A recent breakthrough report from Japan described generation of a bioengineered 3D integumentary organ system from iPS cells. Th is integumentary organ system was capable of producing appendages such as hair follicles and sebaceous glands, and was fully functional upon transplantation into immunocompromised Nude mice (Takagi et al., 2016).

1.2 Hair follicle as an ectodermal appendage Hair follicles are notable appendages of the epidermis (Fig. 1). Th e hair follicle is an evolutionary innovation of the mammals and one of their taxonomical trait. Most mammals have a thick hair coat and devote much of their time to taking care of it for warmth and protection (Fuchs, 2016). Except that, hairs are essential for sensation, camoufl age, dispersion of sebum and pheromones, visual communication and sexual dimorphism (Geyfman et al., 2014). Hairs can provide an example of a unique symbiosis: in sloth, hair groves and cracks are settled by a green algae that can exist only as a sloth symbiont, producing the only green animal on Earth (Suutarila et al., 2010). 1 Review of the literature

Hair follicles are one of the most abundant skin appendages of the body. Due to their accessibility, number and continuous regeneration in adulthood, hair follicles are a great model to study key questions of SC biology. Hair follicle SCs are among the few SCs that can be purifi ed in suffi cient quantities for conducting diff erent types of analysis, such as colony formation assays, transcription profi ling, ChIP-sequencing in vivo, etc (Folgueras et al., 2013).

Sebaceous gland Nerve fiber

Bulge

Permanent part of the hair follicle Arrector pili muscle

Cycling part of the hair follicle Outer root sheath (ORS)

Hair shaft Dermal cell

Matrix

Dermal papilla

Figure 1. Fully formed hair follicle.

1.2.1 Mouse hair types Induction of pelage hair follicles in mice happens in three consecutive waves, giving rise to the four diff erent types of hair shaft s (Dry, 1926; Schmidt-Ullrich and Paus, 2005; Duverger and Morasso, 2009, Ahn, 2015). Th e fi rst wave of induction initiates at E13.5 and forms guard hairs, the longest and the rarest in the coat, which represent from 0.4% to 2-4 % of the hairs depending on the mouse strain (Biggs, Mikkola, 2014; Enshell-Seijff ers et al, 2010; Duverger and Morasso, 2009; Fliniaux et al., 2008; Mustonen et al., 2004). Th e second wave of induction at E16.5 gives rise to the thickest hairs, the awl and auchene, which in the literature are oft en referred to as awl type, and form about 30% of the coat. Th e last wave starts at E18.5 and creates thin, bended zigzag hairs. Some zigzag hairs are also born during the second wave (Chi et al., 2015). Zigzag hairs are the most abundant hairs in the coat (about 70% of the hairs). Th ey form the undercoat fur and provide thermoisolation (Schlake, 2007). Th e type of the hair which is produced by the follicle can change; that happens when hair follicle starts to generate a new hair shaft postnatally. Th is is a feature of that minority of zigzag hair follicles (13%) that arose during the second wave of hair development (Chi et al., 2013).

2 Review of the literature

Early-born zigzag hairs switch to production of the larger awl hairs. It is believed that increase in number of producing cells in the dermal papilla (DP) drives this change. Guard and late- born zigzag follicles do not switch hair type (Chi et al., 2015). Human hair types also change during life. Th e fi rst lanugo hairs are formed in fetal life and are fi ne, brittle and lightly pigmented. Vellus hair replaces lanugo hair in late gestation, and is characteristically short and thin as well. On the scalp, vellus hair is replaced by pigmented terminal hair before or shortly aft er birth. Vellus hair is present throughout childhood and is replaced by terminal hair during or aft er puberty in certain body parts (Duverger and Morasso, 2014).

1.2.2. Morphological stages of hair follicle development Hair follicles appear already during embryogenesis. In mice they are marked at embryonic day (E) 14.5 with epithelial placode and underlying condensate of mesenchymal cells. Directional cell migration and compaction are the main cellular mechanism of hair placode initiation (Ahtiainen et al., 2014). Th e mesenchymal condensate matures into the DP, a specialised mesenchymal population of the hair follicle which regulates multiple aspects of epithelial follicle cell behaviour (Sennett and Rendl, 2012; Biggs and Mikkola, 2014; Morgan, 2014). Along with hair follicle diff erentiation, embryonic epithelium between the placodes diff erentiates into interfollicular epithelium. Th e growing hair placode forms a bud called the hair germ (Fig. 2). At the next stage, the hair peg, the dermal condensate is engulfed by elongating epithelial structures and becomes the DP (Schneider et al., 2009). A rapid growth of the hair follicle is enabled by the population of transit amplifying (TA) cells, or matrix (Mx), formed at the lower part of the growing follicle, in contact with the DP. At the same time the key compartment for hair renewal, the –bulge, begins to specify in the upper third of the hair follicle (Nowak et al., 2008).

Hair placode Hair germ Hair peg Bulbous peg

Specifying bulge Dermal condensate Basal membrane

Dermal papilla

Sox9 Lhx2 Differentiating hair shaft Lhx2 + Sox9

Figure 2. Hair follicle morphogenesis. Cell population expressing diff erent stem cell markers are illustrated.

3 Review of the literature

At the next stage, bulbous peg, all of the hair follicle cell lineages become specifi ed (Fig. 2). Lineage specifi cation has been studied at the renewing postnatal hair follicle (Legue, 2010; Rompolas and Greco, 2014). Since embryonic hair follicle growth resembles in many ways postnatal hair regeneration, this knowledge could be applied to some extent to embryonic morphogenesis as well. Mx cells arrange themselves around the DP in a ring-like manner and each of these precursors become restricted and generate a single diff erentiated lineage of cells that are expanded as a sole column parallel to the long axis of growth (Fig. 3). Th e diff erentiated cells produced collectively by the Mx population form i) the companion layer; ii) three layers of the inner root sheath (IRS): Henle’s and Huxley’s and cuticle layers; and iii) three layers of the hair shaft : hair cuticle, cortex and medulla. Th e IRS cuticle cells mesh like gears with the hair shaft cuticle cells, but near the skin surface, the IRS degenerates to liberate the hair. Th ese Mx-derived layers are enveloped by the outer root sheath (ORS) (Sennett and Rendl, 2012; Rompolas and Greco, 2014), which is contiguous with the basal layer of the interfollicular epidermis. All of these consecutive layers of diff erentiated cells are best visualized at the moment of their specifi cation in the long growing hair follicle. Th e whole process of hair follicle development from the placode until the fully-formed hair follicle is defi ned as hair follicle morphogenesis (Fig. 2). Morphogenesis is completed when the follicle reaches its maximal length. Normally, all hair follicles are born during embryogenesis, however, de novo follicle formation might occur in the adult skin on the re-epithelialized areas of large ( >1 cm in diameter) full-thickness wounds. Hair shaft Th ese large wounds induce de IRS novo the embryonic program of morphogenesis leading to initiation and development of the hair follicles in a similar manner as during Henle’s layer Henle’s layer Huxley’s IRS Cuticle hair shaft Cuticle ORS layer Companion Cortex Medulla embryogenesis (Ito et al., 2007).

K6 AE15 AE13 AE13 AE15 GATA3 Sox9, Lhx2 GATA3, AE15

DP

Matrix Figure 3. Scheme of hair follicle cell lineages at the stage of their diff erentiation in the growing follicle. Immunohistochemical markers are shown for each cell layer.

4 Review of the literature 1.2.3 The hair cycle Hair follicle is one of the few organs that is able to self-renew in repetitive cycles of regeneration called the hair cycle. Two compartments can be distinguished in the hair follicle: a lower cycling part and the upper permanent part (Fig. 1). Th e lower part is renewed in every hair cycle, going fi rst through apoptotic regression (catagen), followed by a rest stage (telogen), and a new growth phase (anagen) (Paus et al., 1999) (Fig. 4). In each cycle, the lower portion is regenerated via a morphogenetic process reminiscent of the embryonic/early postnatal downgrowth of the follicle (Sennett and Rendl, 2012; Rompolas and Greco, 2014). As a result, a new hair follicle is built up at the end of each hair cycle. In mice, the old follicle which gave rise to the new one normally stays at place and keeps its “old” hair, called club hair, for up to three hair cycles until it is shed. At the end, up to three hair follicles with a shared opening may coexist, providing thicker hair coat than it was as a juvenile. In the case in which there are two hair follicles, the founder for the new follicle is always the one which was formed the latest. In normal homeostasis, only this follicle retains the potential to produce new tissue (Fuchs, 2016).

Telogen (rest)

Epithelium Dermal papilla

Club hair

Catagen (regression)

Early anagen (growth)

Late anagen (growth)

Middle anagen (growth)

Figure 4. Hair cycle. (Modifi ed from Rompolas et al., 2013) 5 Review of the literature

In mice, the fi rst two hair cycles are synchronized in the back skin and occur with a relatively precise timing (Müller-Röver et al., 2001). Hair follicle morphogenesis is fi nalized at around postnatal day 10 (P10) with a fully formed hair follicle of the maximal length. Aft er the catagen stage, the hair follicle ends up by P19-P20 in the fi rst telogen, which is very short and lasts for 2-3 days. Aft er that, the fi rst anagen is initiated and lasts for about two weeks followed again by catagen. Th e second telogen is long and lasts from weeks 7 to 12 aft er birth. Th e next hair cycles are progressively less synchronized, meaning that patches of hairs undergo cycling in their own pace. Nearby hair follicles organise themselves into a hair-cycle domain, a region of skin that contains a population of follicles cycling in coordination (Müller-Röver et al., 2001; Plikus et al., 2008). Th e time spent in anagen and catagen does not vary throughout life span, but all telogens aft er the fi rst one are normally long and last for several weeks. Th e precise timing of the fi rst two hair follicle cycles off ers clear time windows for experimental manipulation to study the hair follicle. To analyse changes in anagen onset it is convenient to take advantage of the long, but still synchronized second telogen (Stenn and Paus, 2001). Th e eff ect on hair regeneration can then be analysed at the onset of the second anagen. However, in this case, the exact day of initiation of the hair regrowth cannot be judged precisely. In order to synchronise multiple hair follicle for the activation and to follow hair regeneration step-by-step, hair follicles can be forced to regrow by wax depilation. Aft er depilation, regeneration starts immediately in all the follicles on the depilated area (Hsu et al., 2011), which allows analysis of the quick changes leading to activation of the hair follicle, a time-point which is diffi cult to guess in intact hair cycle. Post-depilated hair follicles progress through the hair cycle synchronously. Even those hair follicles which escaped depilation, but are close to depilated neighbours, start to regenerate via quorum sensing. Th is signal is provided by TNF-α released by macrophages attracted to the depilation-injured skin (Chen et al., 2015). On the naked skin of pigmented mice (such as C57Bl/6), the beginning of the regrowth can be visualised by skin pigmentation due to melanocyte activation. Pigment is produced by melanocytes that have migrated into Mx and is transferred into the hair fi bre. Pigmentation is heaviest from mid to late anagen with little to no pigmentation in telogen (Stenn and Paus, 2001). Th e same growth and regression of the hair follicle occurs in mouse and human. In addition to the three phases described above, human hair has a shedding exogen phase (Higgins et al., 2009). As a result, the human hair follicle never has a club hair and always contains only one hair shaft . Th e length of the stages is what distinguishes human hairs. Scalp hairs are in anagen phase up to 6 years, while the catagen and telogen last only 2-3 weeks and 3 months, respectively (Duverger and Morasso, 2014). In contrast, the mouse hair follicle persists in telogen most of the time (Stenn and Paus, 2001).

1.2.4 Telogen hair follicle Telogen, as described above, is the stage that follows catagen and precedes anagen, and is the longest phase of the mouse hair cycle (Geyfman et al., 2014). At telogen the hair follicle is the shortest in length. Th e telogen hair follicle consists of several morphologically distinct regions (Fig. 5). Th e upper most region at the border between ORS and the basal layer of interfollicular epidermis is the infundibulum, separated by a junctional zone from the isthmus. Th e lower part of the ORS around the base of the club hair forms a bulge. ORS terminates just beneath the bulge

6 Review of the literature into a secondary hair germ, which is a small ball of about 30 cells right above the DP (Greco et al., 2009). Th e term secondary hair germ, oft en shortened to hair germ, refl ects its behaviour in hair regeneration when it acts similarly to the hair germ in embryonic hair follicle morphogenesis. Th e bulge and the hair germ are the compartments that harbour hair follicle SCs, which ensure consequent cycles of hair regeneration (Cotsarelis et al., 1990, Greco et al., 2009). If two populations of SCs exist in the same organ, they can be expected to act in diff erent ways. Th e bulge and the hair germ do indeed possess diff erent functions and produce distinct progeny. At the onset of anagen, hair germ SCs are the fi rst to be activated and to proliferate. Th ey generate Mx, which gives rise to hair follicle structures in a similar way as during hair morphogenesis (Greco et al., 2009; Hsu et al., 2011; Legue and Nicolas, 2005; Rompolas et al., 2015). Th e lower bulge SCs start to proliferate 1-2 days later than the hair germ as the result of a positive Sonic hedgehog signaling loop from the nascent TA cells (Greco et al., 2009; Hsu et al., 2014). Th e bulge progeny builds up the ORS (Rompolas et al., 2013; Hsu et al., 2014). Fluorescence activated cell sorting (FACS) analysis and tracing of the hair follicle SCs at the telogen-to-anagen transition suggests that, in addition to ORS generation, bulge proliferation ensures its self-renewal to compensate for the cells used for the ongoing regeneration. Prior to the initiation of the next growth, there is a cell infl ux from the lower bulge to the hair germ in order to increase the cell population which initiates hair regeneration (Zhang et al., 2009). However, live-imaging of regenerating hair follicle did not register any bulge cells coming out at the anagen onset (Rompolas et al., 2013).

Hair shaft Interfollicular epidermis

Infundibulum Junctional zone Basement memebrane Istmus Sebaceous gland

Bulge Blimp1

Gli1

Hair germ

CD34, Nfatc1

Gli1 Dermal papilla Lgr5 ,-cadherin Sox9, Lhx2 P

Figure 5. Expression of cell population markers in telogen hair follicle. (Modifi ed from Rompolas and Greco, 2014)

A new bulge and hair germ originate at the end of catagen. While all hair germ progeny and most ORS cells die, some slow-cycling ORS cells closest to the bulge survive and contribute to the hair germ and the “new” bulge (Hsu et al., 2011; Rompolas et al., 2013). Th us the bulge makes a niche for the most quiescent SCs which are used economically in the hair cycle, while the whole hair germ undergoes diff erentiation.

7 Review of the literature

Under normal homeostasis, hair follicle SCs contribute exclusively to the hair follicle structures. Th e epidermis is maintained by a separate population of epidermal SCs scattered though the basal layer of the interfollicular epidermis (Kretzschmar and Watt, 2014; Hsu et al., 2014). Upon injury, hair follicle SCs display transient plasticity. Both in superfi cial and full- thickness wounds, bulge and infundibulum SCs migrate upward, proliferate and participate in the epidermal repair process (Levy et al., 2005; Ito et al., 2007; Nowak et al., 2008; Page et al., 2013). Migrating SCs lose their hair follicle identity and adopt interfollicular epidermal fate. However, their progeny do not persist long in the epidermis and are gradually substituted by epidermis-derived cells (Paus et al., 2012; Schepeler et al., 2014; Blainpain and Fuchs, 2014).

1.3 Molecular regulaƟ on of hair follicle development and renewal Hair development and renewal is regulated by the same set of signalling pathways that control the development of the whole organism. Th e most relevant for hair follicle biology are Wnts, Sonic hedgehog (Shh), Fgfs, BMPs and activin of the TGFβ superfamily, and TNF superfamily member Ectodysplasin (Eda).

1.3.1 Wnt pathway Human and mouse have 19 Wnt ligands, which can activate three types of cell responses: the canonical Wnt pathway, the non-canonical Wnt/PCP and the Wnt/Calcium pathways. Among those, canonical Wnt signaling is the most signifi cant and the most studied in the hair follicle. It is mediated by β- which becomes stabilized upon activation of the receptor by binding with Wnt ligand. Frizzled is a principal Wnt receptor which recruits co-receptors for signal transduction (Niehrs, 2012). Stabilised β-catenin escapes from the destruction complex, which consists of Axin, APC (adenomatosis polyposis coli) and other proteins. β-catenin drives gene transcription in the nucleus through binding to Tcf/Lef-1 family of transcription factors (Clevers,et al., 2014). In the course of hair placode development, Wnt/β-catenin signaling is the fi rst to be activated throughout the dermis followed by restricted Wnt10a/b expression in the emerging placode (Zhang et al., 2009). Wnt activates expression of its own inhibitors, Dkk1 in the dermis and Dkk4 in the epithelium (Andl et al., 2002; Bazzi et al., 2007; Fliniaux et al., 2008). Several lines of research have shown the absolute requirement of Wnt signaling for hair development (Table 1). Downregulation of Wnt pathway by ablation of epithelial β-catenin or overexpression of Dkk1 results in complete block of initiation of hair development (Huelsken et al., 2001; Andl et al., 2002; Zhang et al., 2009). Lack of epithelial Wntless, a gene required for Wnt synthesis, blocks hair placode induction in embryogenesis and wound induced hair neogenesis in adult skin (Chen et al., 2012; Myung et al., 2013). Overactivation of Wnt in the epithelium through β-catenin stabilisation results in precocious and excessive induction of hair follicles (Närhi et al., 2008; Zhang et al., 2008). It has also been proposed that dermal Wnt/β-catenin signalling is essential for hair placode formation (Table 1). Deletion of dermal β-catenin prior to hair follicle development with En1-Cre leads to loss of all signs of hair formation, including the dermal condensate, while stabilization of β-catenin with HoxB6-Cre driver leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates (Chen et al., 2012). However, since Wnt signaling is

8 Review of the literature active in the dermis as well, these could be secondary events of β-catenin function in fi broblast diff erentiation. Later ablation of β-catenin in the forming dermal condensate results in arrest of primary hair morphogenesis and loss of several dermal condensate markers indicating a role of activated dermal Wnt in survival or specifi cation of the dermal condensate (Tsai et al., 2014). Activation of canonical Wnt signalling is critical for entry into anagen (Table 1). In late telogen, prior to the telogen-to-anagen transition, DP cells upregulate Wnt signalling genes (Enshell-Seijff ers et al., 2010; Lien et al., 2014). Next, hair germ cells activate Wnt and start to proliferate marking the beginning of anagen (Das Gupta and Fuchs, 1999; Greco et al., 2009). In mouse models, stabilised Wnt/β-catenin activity in epidermis promotes de novo hair follicle formation and drives anagen entry (Gat et al., 1998; Van Mater et al., 2003). In contrast, conditional ablation of β-catenin in hair follicle SCs stopped anagen re-entry (Lien et al., 2014). Wnt co-receptors Lgr5 and Lgr6 are expressed in diff erent, but overlapping populations of SCs, which are able to generate all lineages of the hair follicle (Jaks et al., 2008; Snippert et al., 2010). Lgr5 and Lgr6 bind Wnt agonist R-spondin. Injection of R-spondin facilitates telogen to anagen transition (Li et al., 2016). Anagen progression also depends on Wnt signaling. Mx cells express high levels of Lef-1 (DasGupta and Fuchs, 1999) and epithelial Wnt/β-catenin is required for their proliferation (Choi et al., 2013). Mice with conditional ablation of Wnt7b displayed shortened anagen and shortened hair follicles (Kandyba and Kobeilak, 2014). Hair growth is sustained by continuous signalling activity from the DP supporting Wnt expression. Activation of β-catenin is suffi cient for hair growth even when the DP is laser-ablated (Deschene et al., 2014).

1.3.2 Bmp pathway Canonical Bmp pathway signals through Bmp receptors (Bmpr) I and II, followed by phosphorylation of transcription factors Smad1/5/8 which then bind to Smad4 to activate gene expression. Th e noncanonical Bmp pathway is transduced trough the MAPK cascade. Gold standard targets of canonical Bmp are the group of transcription factors Gata and Inhibitors of DNA binding (Id) proteins. Bmp inhibitors are another essential component of the signaling network as they modulate powerful Bmp signals by blocking ligand binding to the receptor. Among the famous trio of Bmp inhibitors, Chordin, Noggin, and Follistatin (Gilbert, 2014), the latter two are the most important for the skin (Botchkarev, 2003). In skin, the Wnt pathway is tightly coupled with Bmp signalling – their dynamic balance controls many events of hair follicle initiation and regeneration. While Wnt provides a promoting signal, Bmp in general has an inhibitory impact on hair follicle development and regeneration, yet keeping a delicate balance for the tissue activation (Ahn, 2015; Plikus et al., 2008). Expression of the Bmp pathway components in the developing hair follicle is complex and dynamic. Bmp4 and Noggin are expressed in the dermal condensate, while Bmpr1a is expressed in the placode epithelium (Botchkarev et al., 1999). In telogen skin, Bmp2 is broadly expressed in the dermis, while Bmp4 is focused in the dermis close to the bulge (Plikus et al., 2008). Noggin is upregulated in the hair germ and DP at the onset of the hair growth (Plikus et al., 2008; Greco et al., 2009). Due to the complex expression pattern, functional studies of the Bmp pathway in mice were done mostly by ablating Bmpr1a or Smads and altering the level of Noggin, not by aff ecting expression of the ligands (Table 1). Double KO for Smad1 and Smad5 in skin epithelium results in retarded hair morphogenesis and failure to diff erentiate into visible hair (Kandyba et al.,

9 Review of the literature

2013). Noggin defi ciency brings a signifi cant delay in hair placode induction, the eff ect explained by the ability of Noggin to neutralize Bmp4 for hair growth promotion (Botchkarev et al., 1999). Overexpression of Noggin in the epidermis resulted in thickening of epithelium, increased hair density and altered hair types (Plikus et al., 2004). Generally, for hair follicle initiation, Bmp activity needs to be downregulated by Noggin, which results in induction of Lef-1 expression and maintenance of Wnt signalling in the hair germ cells (Jamora et al., 2003; Andl et al., 2004; Samuelov et al., 2015). At later stages of hair morphogenesis, Bmp regulates diff erentiation of the IRS, which was shown in diff erent mouse models with altered Bmp signaling. Downregulation of Bmp signaling by Msx2-driven Noggin overexpression in Mx and hair shaft precursors caused impaired diff erentiation of the IRS and hair shaft (Kulessa et al., 2000), due to the slower migration of diff erentiating hair shaft progenitors (Clavel et al., 2012). Studies of Bmbpr1a cKO mice targeted by Keratin14(K14)-Cre showed that aft er hair follicle formation, Bmp signalling is essential for development of the hair shaft (Kobielak et al., 2003; Andl et al., 2004). Hair follicles lacking Gata3 are not able to generate the IRS (Kaufman et al., 2003) and phenotypically resemble Bmpr1a- defi cient follicles, which also lack Gata3, underlining the importance of Bmp for the terminal diff erentiation of the follicle cells towards IRS and hair shaft (Kobeliak et al., 2003, Genander et al., 2014). In the postnatal hair follicle, Bmp keeps SC in a quiescent, non-activated state. Conditional ablation of Bmpr1a aff ects SC quiescence and leads to transformation of the hair follicles into long-lived tumour-like cysts which are positive for SC markers (Kobielak et al., 2007). In the absence of downstream components of Bmp pathway (double Smad1 and Smad5 KO mice) the formation of the pre-bulge and the bulge, homing quiescent SCs is precluded, and hair follicles remain in prolonged anagen (Kandyba et al., 2013). Th e source of a certain Bmp, Bmp6, in the telogen hair follicle is the K6+ companion layer around the club hair. Bmp6 prevents SC activation, inhibiting regeneration. Depilation removes the club hair together with the attached K6+ cells and takes away the source of the BMP6, releasing hair follicle SCs from inhibition and promoting regeneration (Hsu et al., 2014). Bmp is responsible for modulating the activation signal for hair regeneration in a large- scale manner, driving coordinated hair growth throughout the skin. During telogen, Bmp2 and Bmp4 show periodic expression in the dermis, dividing telogen into two stages with respect to competence for hair follicle regeneration (Plikus et al., 2008). Th e fi rst one is called refractory telogen, when hair follicles persist in a dormant state and are not capable to initiate growth due to high levels of Bmp2 and Bmp4. Decreased dermal Bmp levels defi nes the beginning of hair follicle competence to enter telogen (Plikus et al., 2008). Noggin upregulation and Wnt/β-catenin activation in the hair germ manifest the telogen-to-anagen transition, namely the beginning of regeneration (Botchkarev et al., 1999; Greco et al., 2009; Geyfman et al., 2014). Th us, balancing the Wnt/Bmp level through Bmp inhibitor Noggin controls quiescent and active state of the hair follicle SCs and their competence for hair regrowth.

10 Review of the literature

Table 1. Selected mouse models with altered developmental signaling pathways (OE=overexpression; -/- refers to gene KO in all tissues).

Gene modifi cation (tar- Function of the Phenotype Reference geted tissue) modifi ed gene Wnt pathway K14-Cre mediated abla- Wnt pathway eff ec- Abrogated hair follicle morpho- Huelsken et al., tion of β-catenin tor, cell-cell adhesion genesis, block of hair cycle 2001 (Epithelium) molecule K5-rtTA mediated abla- Wnt pathway eff ec- Block of plucking-induced and Choi et al., 2013 tion of β-catenin tor, cell-cell adhesion spontaneous anagen (Epithelium) molecule K15-CrePGR mediated Wnt pathway eff ec- Graft ed β-catenin defi cient bulge Lien et al., 2014 ablation of β-catenin tor, cell-cell adhesion cells do not form hair follicles (Epithelium) molecule En1-Cre mediated abla- Wnt pathway eff ec- Loss of patterned upregulation Chen et al., 2012 tion of β-catenin tor, cell-cell adhesion of epidermal β-catenin activity (Mesenchyme) molecule and Edar expression Tbx18-Cre mediated abla- Wnt pathway eff ec- Arrest of primary hair morpho- Tsai et al., 2014 tion of β-catenin tor, cell-cell adhesion genesis (Dermal condensate) molecule K14-CreER and K15-Cre- Transmembrane pro- Block of hair placode induction Myung et al., 2013 PR1 mediated ablation of tein required by Wnt in embryogenesis and block of Wntless ligand synthesis wound induced hair neogenesis (Epithelium) K14-Cre and K15-CrePR Wnt ligand Delayed hair follicle activation, Kandyba et al., mediated ablation of shortened anagen and shortened 2014 Wnt7b hair follicles (Epithelium) Sostdc1-/- Modulator of Wnt Enlarged hair placodes Närhi et al., 2012 signaling Lef-/- Wnt pathway tran- No hairs Van Genderen et scription factor al, 1994 Lrp4-/- Wnt co-receptor Retarded hair placode formation Ahn et al., 2014 K14-Dkk1 Wnt pathway eff ec- Complete block of initiation of Andl et al., 2002 (Epithelium) tor, cell-cell adhesion hair development molecule K5-rtTA mediated Dkk1 Wnt antagonist, Block of plucking-induced and Choi et al., 2013 OE binds to Lrp6 Wnt spontaneous anagen (Epithelium) co-receptor K14-Cre mediated stabili- Wnt pathway eff ec- De novo hair follicle morpho- Gat et al., 1998 zation of β-catenin tor, cell-cell adhesion genesis in the interfollicular (Epithelium) molecule epithelium K5-Cre mediated stabiliza- Wnt pathway eff ec- Precocious anagen entry, in- Van Mater et al., tion of β-catenin tor, cell-cell adhesion creased cell proliferation 2003 (Epithelium) molecule K14-Cre mediated stabili- Wnt pathway eff ec- Precocious and excessive induc- Närhi et al., 2008 zation of β-catenin tor, cell-cell adhesion tion of hair follicles (Epithelium) molecule HoxB6Cre-ER mediated Wnt pathway eff ec- Th ickened dermis, enlarged Chen et al., 2012 stabilization of β-catenin tor, cell-cell adhesion epidermal placodes and dermal (Mesenchyme) molecule condensates, prematurely diff er- entiated enlarged hair follicles.

11 Review of the literature

Gene modifi cation (target- Function of the Phenotype Reference ed tissue) modifi ed gene Bmp pathway K14-Cre mediated ablation BMP receptor Compromised diff erentiation of Kobielak et al., 2003 of BMPR1a IRS, downregulation of β-catenin (Epithelium) regulated genes, including keratin genes K14Cre-tm mediated abla- BMP receptor Loss of quiescent SCs. Hair fol- Kobeilak et al., 2007 tion of BMPR1a licles transform into tumor-like (Epithelium) branches K15-CrePGR mediated BMP receptor Hair follicles formed cysts com- Genander et al, ablation of Bmpr1a in the posed largely of ORS, Mx and 2014 second telogen IRS. Precocious hair follicle SC (Epithelium) activation. K14-Cre mediated ablation BMP pathway tran- Retarded hair morphogenesis and Kanduba et al., of Smad1, Smad5 scription factors failure to diff erentiate into visible 2014 (Epithelium) hair. Precluded formation of the bulge. K15-CrePR mediated abla- BMP pathway tran- Precocious anagen onset, hair Kanduba et al., tion of Smad1, Smad5 scription factors follicles remained in prolonged 2014 (Epithelium) anagen Gata3-/- Bmp pathway tran- No IRS. Kaufman et al., scription factor A phenotype resembled Bmpr1a 2003 defi cient follicles. K15-CrePGR mediated BMP target genes, Failure to regrow hairs in repeti- Genander et al, ablation of Id1 regulated by pSmad tive depilations 2014 (Epithelium) transcription factors Noggin-/- BMP2 and BMP4 Secondary hair induction inhib- Botchkarev et al., antagonist ited, impaired downgrowth of 1999 primary hair follicles, downregu- lated Lef1 and Shh expression. Msx2-Noggin OE Bmp2 and Bmp4 Impaired diff erentiation of IRS Kulessa et al., 2000 (Mesenchyme) antagonist and hair shaft Activin pathway ActivinβA-/- Ligand Abnormal vibrissa formation Matzuk et al., 1995 K14-Cre mediated ablation Activin receptor Hair loss and perturbed hair Qiu et al., 2011 of Actvr1b cycle. Many hair follicles trans- (Mesenchyme) formed into sebaceous glands or formed cysts. Hair follicles with increased number of Lef1+ Mx cells. Eda pathway Eda-/- Ligand No primary hair placode forma- Laurikkala et al., (Tabby) tion 2002 Edar-/- Eda receptor No primary hair placode forma- Headon and Over- (Downless) tion beek, 1999 Ectopic IκBαΔN NFκB super-repres- No primary hair placode forma- Schmidt-Ullrich et sor tion al., 2001 K14-Eda Ligand Precocious formation of enlarged Mustonen et al., (Epithelium) hair placodes 2004

12 Review of the literature

Gene modifi cation (target- Function of the Phenotype Reference ed tissue) modifi ed gene

Shh pathway Shh-/- Ligand Arrest of the follicle growth al- St-Jacque et al., ready aft er dermal condensate 1998; Chiang et al., formation, downregulation of 1999 cyclin D1, D2 Gli2-/- Shh pathway tran- Arrest of the follicle growth Mill et al., 2003 scription factor K15-CrePGR mediated Ligand Decrease of bulge proliferation Hsu et al., 2012 ablation of Shh and Mx proliferation, shortened (Epithelium) hair follicles and smaller hair germs aft er the second cycle Sox9-CreER mediated abla- Shh receptor, activator Decrease of bulge proliferation Hsu et al., 2012 tion of Smo of the Shh pathway and Mx proliferation, resulted in (Hair follicle SCs) shortened hair follicle and smaller hair germs Prx1-Cre mediated ablation Shh receptor, activator Loss of the dermal condensate and Woo et al., 2012 of Smo of the Shh pathway arrest of the follicle growth (Ventral dermis) Sox9-CreER mediated abla- Shh pathway tran- Decrease of proliferation in the Hsu et al., 2012 tion of Gli2 scription factor bulge only, shortened hair follicle (Hair follicle SCs) and smaller hair germs Fgf pathway Fgf10-/- Ligand No eff ect on hair follicles Petiot et al., 2003 Fgf20-/- Ligand No dermal condensate in primary Huh et al., 2013 and secondary hair follicles Fgfr2IIIb-/- Fgf receptor in the Reduced number of hair follicles, Petiot et al., 2003 epithelium retardation of hair growth, loss of hairs with age K5-Cre mediated ablation Fgf receptor in the Loss of skin appendages, keratino- Yang et al., 2010 of Fgfr1IIIb and Fgfr2IIIb epithelium cyte hyperproliferation (Epithelium) K14-Fgf7 Ligand Marked suppression of hair follicle Guo et al., 1993 (Epithelium) morphogenesis, thickening of the epithelium

1.3.3 AcƟ vin pathway Activins are another members of the TGFβ superfamily with a prominent function in hair follicle development and regeneration. Activins are dimeric proteins, formed from fi ve types of subunits, from βA to βE (Philips, 2002). Th e most studied members of the activin subfamily are Activin A (a homodimer of two βA subunits), activin B (βBβB) and activin AB (βAβB). Activins bind to a type II activin receptor (or Acvr2) which drives the formation of a heterodimer with type I activin receptor. Th e signal is transduced through Smads2/3 which then partner with Smad4 (Shav-Tal and Zipori, 2002). Acvr1 is expressed by various types of epithelial cells: interfollicular epidermis, ORS and IRS (Roberts and Barth, 1994). Conditional epithelial deletion of Acvr1b leads to hair loss and perturbed hair cycle (Qiu et al., 2011) (Table 1). Hair follicles transform into sebaceous glands or form cysts, and display an increased number of Lef1+ Mx cells, suggesting that Acvr1b signaling is required for the diff erentiation and regrowth of IRS and hair shaft in adult mice (Qiu et al.,

13 Review of the literature

2011). Not much is known about the hair phenotype of ActivinβA KO mice except that they lack whiskers (Matzuk et al., 1995). Sox9 cKO mice have similar hair abnormalities as Acvr1b cKO and downregulated Activin. Apparently Activin is regulated by Sox9 to sustain stemness of bulge and hair germcells operating through pSmad2 (Kadaja et al., 2014).

1.3.4 Ectodysplasin pathway Another important pathway involved in hair development is the Ectodysplasin (Eda)/NF-κB pathway (Botchkarev and Fessing, 2005; Mikkola, 2009). Eda is a tumor necrosis family (Tnf) ligand that binds to the Eda receptor (Edar). Th is signal is transduced by intracellular adaptor Edaradd, which drives activation of IκB kinase (NEMO), followed by phosphorylation of the inhibitory protein IκB and ultimately, release of NF-κB (Mikkola, 2008). In developing skin, Eda is broadly expressed throughout the epithelium, while Edar is focally expressed in the placode. At E14 Eda expression is downregulated in developing placodes but continues in the interfollicular epidermis; later in downgrowing hair follicles both Eda and Edar transcripts are detected in the hair bulb (Laurikkala et al., 2002). Since both ligand and receptor are expressed only in the epidermis, Eda signaling appears to act as a purely intraepithelial method of communication (Laurikkala et al., 2002; Biggs and Mikkola, 2014). Th e central role of Eda in skin appendage development was recognized aft er identifi cation of Eda mutation as a cause for hair and tooth disorders in humans and multiple animal models (Lefebvre and Mikkola, 2014; see chapter 1.7). Indeed a number of studies suggest that this pathway is important for placode stabilization and patterning (Table 1). K14-promoter driven overexpression of Eda (K14-Eda mice) increases hair placode size (Mustonen et al., 2004). Inactivation of the pathway by natural Eda/Edar mutations or by modifi cation of NF-κB signaling abrogates primary hair placode formation (Laurikkala et al., 2002; Headon and Overbeek, 1999; Schmidt-Ullrich et al., 2001). However these mice form rudimentary pre-placodes meaning that Eda is dispensable for initial placode patterning (Schmidt-Ullrich et al., 2006; Fliniaux et al., 2008). Th is initial placode specifi cation requires Wnt/β-catenin activity, which precedes Eda activation. Th e maintenance and further morphogenesis of the pre-placodes of primary hair follicles depends on Eda. Recent data suggest that Eda/Edar/NF-κB also controls primary placode downgrowth through activation of transcription factor Lhx2. Lhx2, in turn, activates Tgfβ2 which leads to ECM remodelling and E-cadherin downregulation, both of which are required for the placode downgrowth (Tomann et al., 2016). As discussed above, the Eda pathway is downstream of Wnt signaling during early hair induction. Nevertheless, placodal Wnat10a and Wnt10b are likely direct NF-κB targets (Zhang et al., 2009; Voutilainen et al., 2012), reinforcing placodal fate stabilization. Bmp inhibitors are also downstream of Eda/NF-κB (Mou et al., 2006; Pummila et al., 2007). Th is suggests a reaction- diff usion model of epithelial-mesenchymal interactions, where Wnt/β-catenin drives placode formation and activates Edar expression to promote Eda signaling for placode stabilization (Mou et al., 2006; Zhang et al., 2009). Edar activation in turn induces Wnt10a/b, but also Dkk4 (Fliniaux et al., 2008). Dkk4 is thought to diff use laterally to prevent placode induction in the surrounding interfollicular epidermis. Long range diff using Wnt10a/b and Bmp inhibitors are received by the placodal epithelium and stabilize the placode. In postnatal hair follicles, Eda and Edar are expressed during catagen in IRS and ORS. Catagen-associated apoptosis was accelerated in Edar-/- mice and aft er treatment of wild type mice with a fusion protein that inhibits Edar signaling, suggesting that Edar regulates apoptosis 14 Review of the literature in keratinocytes during catagen (Fessing et al., 2006). K14-Eda mice have a prolonged fi rst anagen and longer hairs (Mustonen et al., 2003). Th ese data suggest that in addition to its well- established role in hair follicle morphogenesis, Edar signaling is also involved in hair cycle control. Interestingly, Eda and Edar were expressed in the hair germ at the end of the cycle, vanished from the telogen hair follicle and were activated again in the Mx, ORS and IRS at anagen (Fessing et al., 2006). Th is may indicate that Eda also regulates hair follicle regeneration – an aspect that needs further research but would require a mouse with an inducible Eda or Edar deletion to distinguish from the embryonic consequences of Eda-defi ciency (Biggs and Mikkola, 2014).

1.3.5 Shh pathway Among three mammalian hedgehog proteins, only Sonic hedgehog (Shh) has been implicated in hair morphogenesis and regeneration (Biggs and Mikkola, 2014). Th e components of Shh pathway are concentrated in the primary cilia. Shh binds to and inactivates Patched (Ptch), which inhibits signal transducer Smoothened (Smo). Th ere are two types of Shh receptors, Ptch1 and Ptch2, of which the latter has less Smo inhibition activity in the absence of Shh. In the presence of Shh ligand, Ptch inhibition of Smo is abrogated resulting in the nuclear localization of Gli transcription factors, which are the terminal eff ectors of Shh. In vertebrates, there are three Gli transcription factors (Gli1, Gli2 and Gli3). Gli1 is the only full-length transcriptional activator whereas Gli2 and Gli3 act as either positive or negative regulators as determined by posttranscriptional and posttranslational processing (Rimcus et al., 2016). Shh is a powerful oncogene, as ectopic expression of Shh is suffi cient to induce basal cell carcinoma in mice (Oro et al., 1997). Shh is not detectable at the very fi rst steps of placode specifi cation, but appears later in the epithelium of forming placodes. Th erefore, no expression of Shh or Ptch1 has been detected in primary hair preplacodes in Eda/NF-κB mutant mice (Laurikkala et al., 2002; Schmidt-Ullrich et al., 2001; Tomann et al., 2016). Starting from the hair germ, Shh is expressed in the leading tip, in close contact with the DP. Ptch1 is expressed in the epithelium and the mesenchyme, whereas Ptch2 is expressed solely in the epithelium suggesting that Shh acts both in autocrine and paracrine manners (St-Jacques et al., 1998; Chiang et al., 1999; Motoyama et al., 1998). Shh is dispensable for hair placode formation because hair morphogenesis proceeds up to bud stage in Shh-null mice (St-Jacques et al., 1998; Chiang et al., 1999) (Table 1). However, Shh is absolutely required for the downgrowth of the follicle, since Shh and Gli2 mutants display arrest of the follicle growth already aft er the stage of dermal condensate formation. Th e phenotype was associated with reduced epithelial proliferation due to downregulation of cyclins D1 and D2, suggesting a role for Shh as an epithelial mitogen (St-Jacques et al., 1998; Chiang et al., 1999; Mill et al., 2003; Hsu et al., 2014). Hair follicle cells with activated Shh signaling from early stages of development give rise to all cell types of the follicle, as shown in lineage tracing experiments using Gli1-CreER (Xu et al., 2015). Ablation of Shh signaling from one of the two skin compartments, dermis or epithelium, helped to place Shh in the epithelial-mesenchymal crosstalk of the developing hair. Th ere was an attempt to eliminate Shh responsiveness specifi cally from dermis or epithelium by removal of primary cilia from one of the compartments. Only dermal-specifi c KO mice had a similar hair phenotype as Shh nulls, indicating that in dermis, cilia are required for the Shh signal

15 Review of the literature reception (Lehman et al., 2009; Croyle et al., 2011). However, the interpretation of this result is challenging since absence of the whole organelle might aff ect other signaling pathways. Prx1– Cre-mediated Smo ablation in the early embryonic dermis drove loss of the dermal condensate in the ventral skin, and arrest of follicle growth reminiscent of Shh null skin (Woo et al., 2012) (Table 1). Noggin, Sox2 and Sox18 were identifi ed as Shh targets, and overexpression of epithelial Noggin partially rescued the dermal Smo KO hair follicle phenotype by increasing the expression of epithelial Shh. Th is suggests that epithelial Shh ligand activates canonical Shh/Smo cascade in the underlying mesenchyme, and the transduced dermal Shh signal fosters DP maturation. At the same time the readout of the dermal Shh signal is a positive Noggin-Shh feedback loop in the epithelium that drives hair follicle morphogenesis (Woo et al., 2012). Th e telogen hair follicle prior to anagen onset, Shh-responding cells expressing Gli1 and Ptch are located in the hair germ and in the upper bulge, wrapped within the sensory nerve. Shh derived from the nerve is thought to activate the Shh pathway in the upper bulge while Noggin does the same for the hair germ (Brownell et al., 2011; Hsu et al., 2014). Shh is an important link in regulation of hair follicle regeneration. While the initial stage of follicle regrowth relies on hair germ activation by upregulation of Wnt and downregulation of Bmp activity, the progress of regeneration depends on Shh. At the initiation of anagen, hair germ cells start to proliferate fi rst, followed by bulge SC proliferation 1-2 days later (Greco et al., 2009). Hsu and co-authors have shown that newly born Mx generates Shh signal, which promotes proliferation of bulge SCs (Hsu et al., 2014). Shh is sensed by the bulge SCs as long as Shh-producing Mx is in close proximity, less than 200 μm. When the Mx moves further away following hair follicle downgrowth and diff erentiation, Shh is not received by the bulge anymore, and proliferation ceases. Th us, with Shh signalling, SC progeny regulates the rate of SC expansion and controls the well-timed termination of proliferation. Mice with Shh, Gli2 and Smo cKO all have shorter hair follicles and defects in bulge SC proliferation, noticeable aft er the second hair cycle post ablation, since bulge descendants contribute to ORS and hair germ at the end of the cycle (Hsu et al., 2011) (Table 1). Even though bulge SCs have a specifi c co-receptor for Shh, Gas1, extending their sensing abilities, the stimulating eff ect lowers as their Mx progeny moves down with the follicle growth, and bulge proliferation ceases. Th e second outcome of Mx-secreted Shh is upregulation of Noggin and Fgf7 in the DP that keeps Shh signalling active and promotes proliferation of the Mx cells themselves. Th us embryonic hair development and regeneration in adulthood utilize the same signaling pathways and apply them in a similar context (Hsu et al., 2014).

1.3.6 Fgf pathway Th e Fgf family contains 22 Fgf ligands that signal through 7 isoforms of Fgf receptor tyrosine kinases (Fgfr), encoded by 4 genes. Due to multiple redundancy between the Fgf ligands, the strategy for studying Fgf function focuses on deactivating the receptors of the pathway. Ligand binding drives dimerization and activation of the receptor. Th e signal transduction may go through MAPK, PI3K-Act/PKB, PLCγ and STAT intracellular pathways (Ornitz and Itoh, 2015). In the developing skin, Fgf ligands are mainly expressed in the dermal component. Among the four genes of Fgf receptors, three are expressed in the skin: Fgfr1 mainly in the dermis and Fgfr2 and Fgfr3 mainly in the epidermis (Zhang et al., 2006). Mice lacking IIIb isoform of Fgfr2, Fgfr2IIIb, had reduced number of hair follicles and retardation of hair growth (Petiot et al., 2003)

16 Review of the literature

(Table 1). However, for hair follicle initiation, Fgfr2IIIb needs to be downregulated in placodal epithelium as it transduces signalling that favours interfollicular epidermal diff erentiation (Richardson et al., 2009). Fgf10 ablation drove epidermal hypoplasia but did not aff ect hair follicles (Petiot et al., 2003). Fgf7, also known as keratinocyte growth factor (KGF), has a negative impact on hair morphogenesis, but promotes epithelial diff erentiation, suggested by data from K14-Fgf7 overexpressing mice and Fgf7-treated embryonic skin cultures (Guo et al., 1993; Richardson et al., 2009). Fgf20, expressed in the placodes, is required for the formation of dermal condensate. All the dermal condensate markers of primary and secondary hair follicles are abolished in Fgf20-/- mice, but hair placodes do form meaning that mesenchymal and epidermal morphogenesis are uncoupled at the early stages of hair development (Huh et al., 2013). Interestingly, spontaneous mutation of Fgf20 in chicken leads to a more severe phenotype with loss of almost all body feathers, while Fgf20-defi cient mice lack only guard and some awl/auchene hairs (Huh et al., 2013; Wells et al., 2012; Houghton et al., 2007). Fgfs are involved in regulation of postnatal hair follicle homeostasis as well. Ablation of both Fgfr1IIIb and Fgfr2IIIb leads to impaired hair cycle and loss of hair follicles by fi ve months of age (Yang et al., 2010) (Table 1). Fgfr1 and Fgfr2 are receptors for Fgf7/10 (Zhang et al., 2006). Fgf7/10 come from the DP and provide the proliferation signal received by the hair germ cells, but not by the bulge (Greco et al., 2009). Infrequent bulge proliferation is ensured not only by its further location from the DP, but also by the inhibitory signal from the epithelially produced Fgf18. Fgf18 comes from the cells of K6+ inner bulge layer and keeps hair follicle SCs quiescent providing a high threshold for anagen activation (Hsu et al., 2011).

1.4 Key transcripƟ on factors regulaƟ ng hair follicle stem cells During telogen, many genes display localised expression domains within the follicle and thereby mark distinct cell populations. Moreover, the distribution of many SC markers changes dramatically during the hair cycle. It is under question whether the observed molecular heterogeneity signifi es a functional compartmentalisation of the niche, with the presence of diff erent populations representing strictly defi ned fates during regeneration. Furthermore it is not clear if the expression of the marker genes refl ects the intrinsic diff erentiation potential of the cell or merely its physiological state at a certain position where the cell ends up within the niche (Rompolas and Greco, 2014). Th e most used and studied markers of the hair follicle SCs are Keratin 15, CD34, Wnt coreceptor Lgr5, transcription factors Sox9, Lhx2, Nfatc1, Runx1, Tcf3 (Rompolas and Greco, 2014; Schepeler et al., 2014; Goldstein and Horsley, 2012) (Fig. 5). Th ese markers spread throughout both bulge and hair germ and do not distinguish between two SC populations (Rompolas and Greco, 2014). Very few markers are unique for bulge or hair germ cells: CD34 and Nfatc1 are restricted to the bulge (Trempus et al., 2003; Horsley et al., 2008), whereas P-cadherin and Runx1 are enriched in the hair germ (Muller-Rover et al., 1999; Osorio et al., 2008). In addition to these diff erences in molecular markers, bulge and hair germ cells are also distinguished by diff erences in gene expression signatures, in proliferation potential in vitro and in time of activation for anagen (Greco et al., 2009). Th e cellular source within the ORS for the new bulge and hair germ formed at the end of the hair cycle also diff ers (Hsu et al., 2009; Zhang et al., 2009). All these evidences

17 Review of the literature support the argument that bulge and hair germ represent biochemically and functionally separate populations of hair follicle SCs. At the same time, hair follicle SC niche displays a great level of plasticity, since both bulge and hair germ are able to functionally substitute each other and initiate hair regeneration following physical ablation (Ito et al., 2002; Rompolas et al., 2013). Among SC markers, transcription factors represent the most interesting group as they are at the gate of regulation of diff erent avenues of cell fate specifi cation and lineage choices. Common eff orts of the past and recent studies clarifi ed the role of the key marker transcription factors for hair regeneration and maintenance. In the following chapters, four of these factors, Sox9, Lhx2, Nfatc1 and Runx1, will be described in more detail.

1.4.1 Sox9 – pioneer factor and master gene regulator of hair follicle stem cells Sox9 could be named “the king of transcription factors” regulating hair follicle SCs. Recent studies identifi ed it as a pioneer factor of hair follicle lineage diff erentiation (Adam et al., 2015). Sox9 is expressed in the suprabasal cells of the hair placode marking a distinct cell population negative for other transcription factors such as Lhx2 and Lef-1 (Vidal et al., 2005; Nowak et al., 2008). In the course of hair development, Sox9 is expressed in the upper region of the follicle, marking the “pre-bulge” region and ORS and diminishing towards the Mx (Vidal et al., 2005; Nowak et al., 2008; Kadaja et al., 2014) (Fig. 2). Bulge and hair germ of adult hair follicle are positive for Sox9, whereas in anagen follicle Sox9 expression is silenced once ORS reaches Mx, in the same way as in embryogenesis (Vidal et al., 2005; Nowak et al., 2008; Kadaja et al., 2014; Rompolas and Greco, 2014). In mouse, very few interfollicular epidermal cells are Sox9+ (personal observation). In contrast, in human skin, Sox9 is expressed in epidermal cells (Shi et al., 2013). Cell fate mapping Sox9-Cre::Rosa26R-LacZ on the tail skin showed that the progeny of Sox9+ cells contribute to all cell lineages of the hair follicle, including sebaceous gland, and even were detected in post-scratch interfollicular epidermis (Nowak et al., 2008). Lineage tracing of telogen hair follicles using Sox9-CreER::Rosa26-YFP mice confi rmed multipotency of Sox9+ cells and their long-term potential even 8 months post-labelling (Kadaja et al., 2014). Sox9 ablation at embryogenesis with epithelial specifi c Y10-Cre (not expressed in head and shoulder region) or K14-Cre resulted in complete loss of coat hair (Vidal et al., 2005; Nowak et al., 2008) (Table 2). Sox9 cKO hair follicles were initiated on time, but the SC niche never formed. Proliferation of Mx was aff ected, that resulted in shortened follicles that degenerated by P10-P34 (Vidal et al., 2005; Nowak et al., 2008). Lineage tracing of embryonic SC precursors from the hair peg stage revealed that hair follicle SCs originate from Sox9+ cells (Xu et al., 2015). Th us embryonic Sox9 targeting and lineage tracing evidence that Sox9 is dispensable for hair initiation but is required for the SC specifi cation, and that SCs are established early in embryogenesis. Whether Sox9 is essential also for SC maintenance and whether it is needed for SC activation for regeneration was explored in mice with Sox9 ablation in the adult hair follicle aft er formation of bulge and hair germ (Kadaja et al., 2014). Shaved Sox9 cKO mice had defi cient hair recovery aft er natural activation of the second anagen: soon aft er anagen entry SC markers waned and the bulge accumulated keratinised cysts, indicating a shift to epidermal fate. Postnatal phenotype of Y10-Cre::Sox9fl oxed/fl oxed mimicked embryonic Shh-defi cient models, as hair follicles similarly acquired epidermal characteristics with ectopic expression of Gata3 (Nowak et al., 2008). In addition, Mx size was decreased in all Sox9 defi cient mouse models likely

18 Review of the literature due to poor ability of SCs to replenish ORS and Mx pools, though in adults this change was less prominent. Telogen-to-anagen transition was not aff ected, but once regeneration has started and bulge cells initiate proliferation, they were not able to return to quiescence. Th erefore Sox9 is dispensable for SC activation at regeneration, but suppresses epidermal diff erentiation within the niche. Notch and Wnt were not aff ected by loss of Sox9. Instead, among the pathways preferentially regulated by Sox9, most hits were associated with Activin/Tgfβ (Kadaja et al., 2014). Actvr1b cKO have similar phenotype as Sox9 cKO mice (Qui et al., 2011). Treatment of Sox9 cKO mice with Activin decreased formation of cysts, restored CD34 immunoreactivity of the bulge, increased expression of some SC genes and decreased epidermal signature genes. However, Activin application could not help Sox9-defi cient SCs to return to quiescence aft er proliferation. Th us Sox9 acts on genes of the Activin pathway to sustain stemness and block interfollicular epidermis diff erentiation (Kadaja et al., 2014). Sox9 itself lies downstream of Shh since it is absent in Shh-/- and Gli2-/- mice while upregulated in basal cell carcinoma tissue known for high Shh activity level (Vidal et al., 2005). At early stages of follicle development placode-derived Shh maintains Sox9 expression in the hair peg (Ouspenskaia et al., 2016). Aft er Mx establishment, this population produces Shh and functions to stabilize Sox9. Wnt negatively regulates Sox9 as attenuation of Wnt signaling is a prerequisite for maintenance of Sox9 expression in the developing bulge (Xu et al., 2015).

1.4.2 Lhx2 – a signpost at the matrix/sebocytes crossroads Lhx2 is another powerful regulator of SC fate. It was the fi rst identifi ed marker expressed specifi cally by both embryonic SC precursors and postnatal hair follicle SCs in the bulge (Rhee et al., 2006). Lhx2 expression is initiated aft er placode formation at the leading edge of the hair peg (Rhee et al., 2006; Mardaryev et al., 2011) (Fig. 2). It is likely that Lhx2+ hair germ pocket cells are a transit population with high levels of Wnt activity that do not obtain long-term properties neither generate long living progeny, contributing only to the structures that are all depleted during the fi rst hair cycle (Ouspenskaia et al., 2016). As hair development progresses, Lhx2+ zone spans up to the ORS and mixes with the Sox9 expressing domain. True Lhx2 expressing hair follicle SCs appear from this upper bulge area, coexpressing Lhx2, Sox9 and other hair follicle SC transcription factors (Nowak et al., 2008). Whether these cells are the direct descendants of Lhx2+ hair germ pocket cells, or a separate cell population that gained Lhx2 independently remains unknown. Based on stage-by-stage Lhx2 expression pattern, it is likely that they are progeny of the germ Lhx2+ cells, which attenuate Wnt as they transit into suprabasal layers. Lineage tracing of Lhx2-expressing cells might help to unveil this. However, it is clear that the progeny of prebulge Lhx2+ cells will form SC compartments of the telogen hair follicle, where Lhx2 is expressed in the bulge and secondary hair germ (Nowak et al., 2008; Rhee et al., 2006). In the developing hair placode, Lhx2 is regulated by the Eda/Edar/NFκB signaling axis (Tomann et al., 2016). Downstream of Lhx2 lay hair follicle SC genes Sox9 and Tcf3, as K14- Lhx2 overexpression drives their induction in interfollicular epidermis, suggesting that Lhx2 plays a role in specifi cation of hair follicle SC fate (Rhee et al., 2006) (Table 2). Total Lhx2 null mice are lethal around E15.5-E16.5, however they develop morphologically normal hair follicles, but in 40% reduced number. Graft ed mutant skin developed generally normal hairs, but they

19 Review of the literature were programmed for precocious anagen (Rhee et al., 2006). K14-Cre-driven Lhx2 cKO mice showed the same premature anagen, did not develop a proper SC niche as suggested by the absence of CD34 and Nfatc1 markers, and fi nally became bald. With age, Lhx2-defi cient bulge cells diff erentiate into sebocytes indicating a role for Lhx2 in stabilising SC choice. SCs lacking Lhx2 make a stochastic choice between sebocyte and Mx diff erentiation (Folgueras et al., 2013). Transgenic expression of Lhx2 in postnatal hair follicle drives anagen entry, a phenotype similar to the mouse models with Lhx2 deletion (Törnqvist et al., 2010), suggesting that too much of this transcription factor also causes perturbation in regulation of SC behaviour. To gain insights into the molecular mechanisms of Lhx2 regulation, fi rst genome-wide mapping of Lhx2 binding genes was performed on embryonic skin from Lhx2-/- mice (Mardaryev et al., 2011). Sox9, Tcf4 and Lgr5 were found to be direct Lhx2 targets as their promoters were occupied by Lhx2, and further, Sox9 and Tcf4 reporter plasmids were activated in the presence of Lhx2. Transcription repressors TCF3/4 showed strongest downregulation in Lhx2-defi cient skin, suggesting a link with Wnt signaling as well as a role for Lhx2 in negative regulation of gene expression (Mardaryev et al., 2011). Most Lhx2 targets in the adult skin identifi ed from chromatin profi ling of Lhx2 cKO were diff erent from the embryonic ones (Folgueras et al., 2013), though Wnt-related and hair follicle SC genes were in the list as well. Th e majority of top categories encompassed proteins involved in the and in cell-cell and cell-ECM adhesion. Accordingly, Lhx2 cKO hair follicles revealed severely compromised apico-basal polarity and perturbation in in the SC niche. Lhx2 cKO mice displayed “one-bulge phenotype”: only one bulge and hair shaft were present aft er the second and following hair cycles due to poor anchoring of the club hair eventually leading to its loss. A wild type hair follicle never accommodates more than 4 bulges. Th e fate of the older bulges is still unknown. An intriguing idea is that once a bulge sheds its old hair, its SCs with their low division history fold into the newer bulges and “rejuvenate” the hair follicle SC pool to improve its effi ciency in hair cycling and wound repair (Fuchs, 2016). In Lxh2 cKO impaired cytoskeletal organization of K6+ companion layer aff ected its ability to keep a club hair, which was shed together with K6+ cells. Loss of Fgf18 and Bmp6 inhibitory signals from K6+ cells allowed precocious anagen entry (Folgueras et al., 2013). Lhx2 operates as an important regulator of epithelial SC activity in the skin response to injury (Mardaryev et al., 2011). Wound-induced anagen in the follicles adjacent to the wound was accelerated in Lhx2+/- mice, in agreement with precocious anagen noticed in Lhx2 defi cient mouse models. In the context of wound healing, Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits hair cycling (Mardaryev et al., 2011).

20 Review of the literature

Table 2. Selected mouse models with altered expression of hair follicle SC transcription factors.

Modifi ed gene Phenotype Reference Sox9 Y10-Cre mediated abla- No visible hairs. Severe proliferative defects, SC niche never Vidal et al., 2005 tion of Sox9 formed. Th e ORS acquired epidermal characteristics with ectopic expression of Gata3. K14-Cre mediated abla- Normal hair follicle initiation, no hairs appeared in the adult- Nowak et al., 2008 tion of Sox9 hood. Shortened Mx, bulge never formed, loose of all bulge markers, though Lhx2 persists in the hair peg. K15-CrePGR mediated Poor coat recovery aft er shaving. Telogen-to-anagen tran- Kadaja et al., 2014 ablation of Sox9 in the sition was not aff ected. SCs could not return to quiescence, second telogen undertook epidermal fate. Downgrowth arrest due to failure of ORS production. Lhx2 Lhx2-/- 40% less hair follicles, no SCs formed, precocious anagen. Rhee et al., 2006 K14-Lxh2 No more hairs induced, induction of Sox9 and Tcf3 in inter- Rhee et al., 2006 follicular epidermis. Lhx2-/- and Lhx2+/- Wound re-epithelization was retarded, anagen onset in the Mardaryev et al., follicles located close to the wound was accelerated. 2011 K14-Cre mediated abla- “One-bulge” phenotype: a widespread loss of club hairs. Mice Folgueras et al., tion of Lhx2 became bald with age. Telogen-to-anagen transition was not 2013 aff ected. Transformation of the SC niche into a sebaceous gland. Perturbed architectural organization of the niche, poor club hair anchoring. Nfatc1 Nfatc1-/- Analysis of skin graft phenotype: premature shedding of the Horsley et al., 2008 club hair, precocious entering into anagen K14-Cre mediated abla- No club hair loss, but precocious anagen entry Horsley et al., 2008 tion of Nfatc1 Runx1 K14-Cre mediated abla- Block of SCs in quiescence, impaired hair cycle. Osorio et al., 2008 tion of Runx1 Defective hair follicle SC activation, but not maintenance, proliferation or diff erentiation. β-actin-CreER mediated Impaired anagen induction and hair follicle SC proliferation. Hoi et al., 2010 ablation of Runx1 Reduced rates of tumor formation. Runx1-CreER mediated Delay in hair shaft emergence, fewer hair follicles were Osorio et al., 2011 deletion of Runx1 due formed. Enlarged sebaceous glands, which transformed into to creation of truncated cyst-like looking structures fi lled with sebum that lacked Mx version of Runx1 protein and bulge K14-rtTA mediated Induced catagen. Quiescent bulge SCs acquired hair germ Lee et al., 2014 overexpression of Runx1 fate. Fox transcription factors K14-Cre, K15-CrePR and “One-bulge” phenotype. Hair follicle SCs failed to return to Wang et al., 2016 Foxn1-Cre mediated abla- quiescence and got prematurely activated. tion of Foxc1 “One-bulge” phenotype. Bulge SCs became primed to pro- Lay et al., 2016 K14-Cre mediated abla- liferate, increased colony forming effi ciency in vitro. Conse- tion of Foxc1 quently accelerated hair cycles. Sparse hair coat. Foxn1-/- Hair follicles developed normally. Hair fi bres structure was Mecklenburg et al., aff ected due to downregulation of hair . Mx diff eren- 2004 tiation was altered toward an epidermis-like.

21 Review of the literature 1.4.3 Nfatc1 – quiescence and aging of hair follicle stem cells A prominent role of Nfatc1 in regulation of hair follicle SCs was fi rst suggested by transcriptional profi ling of the bulge cells and hair germ cells (Blanpain et al., 2004; Morris et al., 2004; Tumbar et al., 2004, Rhee et al., 2006). In many cell types, cytoplasmic NFAT (NFATc) proteins are confi ned to the cytoplasm under basal conditions. Upon effl ux of intracellular calcium, Nfatc proteins are translocated to the nucleus to regulate gene transcription (Crabtree and Olson, 2002; Crabtree and Graeb, 2008). Nfatc1 is expressed at the hair germ stage in a rather restricted zone in the prebulge and in the upper bulge at telogen (personal observation; Horsley et al., 2008). Graft ing of skins from germline-deleted Nfatc1 mice revealed precocious anagen entry and loss of the club hair, paralleling the “one bulge” phenotype observed in absence of Lhx2 (Table 2). Precocious anagen entry is typical for this phenotype due to loss Fgf18/BMP6 inhibiting signal. Nfatc1 cKO had a milder though similar phenotype to that of the total nulls. In vitro gain of function experiments confi rmed the role of Nfatc1 in inhibiting keratinocyte proliferation (Horsley et al., 2008). Overall, Nfatc1 acts in the regulation of hair follicle SC quiescence. Inhibition of BMP activity by application of Noggin downregulated Nfatc1, indicating that Nfatc1 is downstream of BMP signaling (Keyes et al., 2013). In turn, through inhibition of cyclin-dependent kinase 4, NFATc1 downregulates proliferation and promotes quiescence. As a biological proof of this concept, Nfatc1 is upregulated in the hair follicle SCs in aged mice. Moreover, Nfatc1 was suggested to be a master regulator of SC quiescence since nearly 40% of the upregulated and 19.5% of downregulated genes of aged hair follicle SCs are bound by Nfatc1 upon activation. Th is suggests that Nfatc1 contributes markedly to the maintenance of the quiescent bulge signature (Keyes et al., 2013).

1.4.4 Runx1 – an acƟ vator of anagen onset Several mouse lines were designed to study the function of Runx1 in skin. Runx1-CreER::Rosa26R mice demonstrated that Runx1 expression starts at about E15.5, aft er follicle initiation and that Runx1-expressing embryonic cells are precursors of adult hair follicle SCs (Raveh et al., 2006; Osorio et al., 2011). Lineage tracing experiments showed that early embryonic Runx1+ epithelial cells are hair follicle progenitors that contribute most substantially to the ORS and are stably maintained during postnatal cycling. Runx1+ epithelial cells of advanced hair follicle contributed substantially to Mx, which generated the fi rst hair shaft and IRS, and were nearly completely lost in postnatal morphogenesis. Early, but not late, Runx1+ mesenchymal cells showed contribution to the upper dermis and to the DP during postnatal morphogenesis (Osorio et al., 2011). In the adult hair follicle Runx1 is enriched in the hair germ (Osorio et al., 2008). Endogenous Runx1 expression is downregulated in the hair germ as it begins proliferation to make Mx at anagen and is upregulated in apoptotic Mx cells. ORS cells that once expressed Runx1 contribute to both ‘‘new’’ bulge and hair germ formation (Lee et al., 2014). Runx1 loss in the early embryonic epithelium in K14Cre::Runx1fl /fl (cKO) mice resulted in impaired timely hair placode formation and hair germ downgrowth and delayed hair morphogenesis, which was largely overcome by birth (Osorio et al., 2011) (Table 2). Hair follicle SCs of cKO mice failed to respond to normal growth activation signals during the initiation of adult hair cycling phase. Two mouse models with distinct spatiotemporal Runx-1 deletion demonstrated that Runx1 epithelial defi ciency delayed anagen onset by weeks, despite

22 Review of the literature the presence of SCs at normal numbers (Osorio et al., 2008; Hoi et al., 2010). Such prolonged telogen is consistent with a role of Runx1 in regulating early stem/progenitor cell fate choice and diff erentiation to hair cell lineages. Runx1 deletion did not aff ect the diff erentiation potential (multipotency) and fate decision of progenitors and hair follicle SCs. Runx1 cKO SCs maintained their long-term potential and repeated stimulation (depilation, photo stimulation and close shaving) did not exhaust the mutant SC pool. Th us, Runx1 is a modulator of keratinocyte proliferation in the hair germ, hair growth and SC activation (Osorio et al., 2008). In doing so, Runx1 operates at least in part through Wnt signaling, since deregulated Lef-1 and Wnt signaling pathway was detected in Runx1 epithelial KO skin (Osorio et al., 2011). Runx1 promotes SC proliferation also through repressing transcription of CDK inhibitors (p21, p27, p57 and p15) (Lee et al., 2013). Th us, Runx1 is needed for normal hair follicle homeostasis at the transition into the adult hair cycling stage, while it is dispensable during injury repair (Osorio et al., 2008). Runx1 is necessary for promoting anagen onset and stem cell activation. Th is was shown by deleting Runx1 aft er the end of morphogenesis with inducible β-actin-CreER (Hoi et al., 2010). Since Runx1 is abundant in the mesenchyme, there was a question if Runx1 might also work from the dermis to regulate the epithelial–mesenchymal cross talk. Runx1CreER targeted Runx1 mainly in the skin mesenchyme and impaired adult hair follicle maintenance. Epithelial Runx1 cKO showed changes in Wnt signaling: expression of the BAT-GAL Wnt reporter was downregulated, activating molecules such as secreted ligands Wnt11 and Wnt2b were downregulated along with the Fzd4 receptor. Th e dermis responded to loss of Runx1 in the epithelium by upregulating Wnt inhibitory molecules Sfrp1, Apc, Gsk3B, and Nlk, and by lowering activating Wnt factors Bcl9 and downstream target Foxn1. Th is suggested that appropriate levels of Runx1 activity might be important for amplifying Wnt signaling in conjunction with the core components of the canonical pathway (Osorio et al., 2011). However, ChIP-sequencing studies are needed for the fi nal confi rmation of the direct regulation of canonical Wnt by Runx1 transcription factor. Th e function of Runx1 in activation of SCs and promoting hair regeneration was confi rmed in gain-of-function experiments (Table 2). Forced Runx1 expression induced catagen and simultaneously promoted changes in the quiescent bulge SC transcriptome toward a cell state resembling the epithelial hair germ fate, namely short-lived progenitors. Th is cell-state transition was functionally reversible suggesting that SC diff erentiation and dediff erentiation are likely to occur during normal follicle degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location in the niche (Lee et al., 2014). Interestingly, in transgenic Runx1 mice, Runx1 was insuffi cient for anagen onset but suffi cient to induce catagen, meaning that the presence of inductive anagen signals, not present in the skin at second telogen, might be required in addition to Runx1 for normal anagen onset. SC diff erentiation into early progenitors (hair germ cells) is probably driven by upregulation of certain fate determinants, of which Runx1 appears to be an early one. Th e early progenitor transition state is metastable, as it would either fall backward from the hill of biological potential to remake SCs (when hair germ progeny from the ORS makes a new bulge) or fall forward to divide and make the TA cells, which are more stable cell states (Osorio et al., 2011).

23 Review of the literature 1.5 CoordinaƟ on of hair follicle stem cell regulaƟ on If we would be brave enough and create an artistic parallel between hair follicle SC dynamics and a music structure, we can associate a mechanism of action of each SC transcription factor or signaling pathway with a melodic phrase. Despite that the consequences of the ablation of each of the key SC transcription factor or signaling pathway have been deeply studied and rather well understood, multiple, thoroughly-structured melodies are still inconherent and do not form a perfect harmony of wise regulation of SCs in the adult organism. However some chords formed by interaction of certain factors already sound clear for us. Th is is mostly relevant to the interaction between Wnt, Shh and Bmp signaling described earlier, and a link between Sox9, Lhx2 and some other transcription factors which will be discussed now. Lhx2 is responsible for the bulge SCs to diff erentiate into Mx cells rather than into sebocytes. Without Lhx2, hair follicle SCs seemed to randomly choose between these two fates. Th is refl ects an instability of the fate choice rather than the absolute defect in lineage selection, confi rmed by the fact that both Wnt-enhancing and -inhibitory genes were among the Lhx2 targets, suggesting that within the confi nes of the niche, the pathway could be misregulated rather than fully suppressed (Folgueras et al., 2013). Lhx2 is one of the genes most markedly repressed upon loss of Sox9, but the loss-of-function phenotypes of these genes are quite distinct from each other. Similarly, both Lhx2 and Sox9 orchestrate quiescence and stemness, and their loss leads to proliferation and loss of hair follicle SC markers and behaviour. However, each has its own impact on the lineage suppression, with Lhx2 playing a more important role in suppressing sebocyte diff erentiation, and Sox9 playing a greater role in supressing epidermal diff erentiation. Together, these factors maintain stemness and direct SCs along the correct lineage. Further, Sox9 binds and regulates Lhx2 in hair follicle SCs, while Lhx2 ChIP-sequencing did not reveal binding to Sox9, suggesting that in this pair, Sox9 is upstream on Lhx2 (Folgueras et al., 2013; Kadaja et al., 2014). It was recently suggested that direction to a certain cell fate choice occurs due to coordinated work of SC-specifi c transcription factors occupying certain areas of chromatin known as super- enhancers (Adam et al., 2015). Super-enhancers are large (>10kb) clusters of active enhancers simultaneously occupied by multiple tissue-specifi c transcription factors (Adam and Fuchs, 2016). In hair follicle SCs, Sox9 is the most frequently bound transcription factor on the super- enhancers that rules transcription of cell fate genes, mostly transcription factors themselves. Other hair follicle SC marker – transcription factors (Lhx2, Nfatc1, Tcf3/4) also were found bound to super-enhancers, but with less frequency than Sox9. Induced Sox9 expression in the epithelium drove activation of other super-enhancers that control hair follicle SC transcription factor genes, and specifi cally PcG-silenced Lhx2 and Tcf7l2 (encodes for Tcf4) genes. Th is indicates that Sox9 is a pioneer factor capable of initiating H3K27 acetylation and thus overriding the silenced chromatin barrier. Th e role of pioneer factors, directing cell lineage choice, is seen as DNA-binding proteins whose levels dictate whether super-enhancers will be epigenetically active or silenced. Sox9 ablation during embryogenesis blocked Lhx2, Tcf7l1 (encodes for Tcf3) and Tcf7l2 expression. Conversely, ectopic Sox9 expression in cultured hair follicle SCs induced Lhx2 >80x, Tcf7l1 and Tcf7l2. Neither Sox9, Tcf7l1 nor Tcf7l2 showed such sensitivity to Lhx2, indicating a special importance for Sox9 in regulating SC super-enhancer activity and putting Sox9 at the top in the hierarchy of SC transcription factors (Adam et al., 2015).

24 Review of the literature

Adult hair follicle SC behaviour is determined by the inputs from the multiple niche cell types, including SCs themselves. Th is guarantees that the decision of when to make tissue or which lineage to follow happens in synchrony and only when a variety of cellular inputs from the tissue are met (Kadaja et al., 2014).

1.6 Hair follicle stem cell speciĮ caƟ on In contrast to the thoroughly studied homeostasis of the adult hair follicle SC niche, its establishment during development is relatively poorly understood. Th e hair follicle SC niche begins to be established already during embryonic life. Segregation of the cell population expressing SC markers is clear from the hair germ stage: the leading edge cells are positive for Lhx2, while the suprabasal population expresses Sox9 (Rhee et al., 2006; Nowak et al., 2008) (Fig. 2). Th e functional signifi cance of cellular heterogeneity of the emerging hair follicle have become clear recently. Wnt/β-catenin pathway is vitally required for the formation of hair follicle SCs (Huelsken et al., 2001), but this eff ect seems to be indirect. From the hair germ stage, a zone of attenuated Wnt signaling emerges in the upper hair follicle (Xu et al., 2015). It has been proposed that these cells originate from the earliest divisions within the placode, which are almost exclusively perpendicular to the basement membrane (Ouspenskaia et al., 2016). Th e daughter cells displaced from the basement membrane downregulate Wnt signaling and become Wntlo, Sox9+ cells. From this stage onward, basal cells with activated Wnt signaling, Wnthi cells, stop proliferation and do not contribute to the formation of hair follicle SCs (Ouspenskaia et al., 2016). Th e fate of Wnthi and Wntlo progenitors was followed by Axin2+ and Sox9+ lineage tracing, respectively (Xu et al., 2015). Only about 15% of hair follicle SCs originate from Wnt-expressing cells, and those are lost aft er the fi rst hair cycle. On the contrary, Sox9+ hair peg precursor cells are the origin of the most hair follicle SCs, which persist and expand aft er one round of hair regeneration to become essentially all of the SCs (Xu et al., 2015). Shh is initially expressed in the placode and at the germ stage. Shh+ cells are restricted to the bottom of the germ pocket, i.e. to the basal Wnthi cells (Ouspenskaia et al., 2016). Shh-expressing placode and hair germ cells give rise to all cell types of the hair follicle, including the bulge (Levy et al, 2005; Xu et al., 2015). Recent lineage-tracing data suggest that soon aft er, in the hair peg, Shh-expressing cells restrict their diff erentiating potential, lose the ability to form SCs and mostly contribute to diff erentiated lineages. In both Shh-/- and Smo cKO hair follicles, the population of Wnt hi cells was expanded while Wntlo, Sox9+ population decreased, suggesting that Shh activity downregulates Wnt signalling and promotes specifi cation of Wntlo Sox9+ cells (Ouspenskaia et al., 2016). Hair follicle SCs originate from Wntlo progenitor cells from the hair peg that loose Shh expression but instead respond to Shh. Th ese cells gain the expression of adult SC markers (Xu et al., 2015), such as one of the earliest markers Sox9, which is known to depend on Shh (Vidal et al., 2005).

25 Review of the literature

Th e link between activated Wnt signaling and Sox9 expression was studied independently using mice with stabilized β-catenin under Nfatc1-CreER and mice with lentivirus-induced expression of APC protein. It was proved that elevated Wnt signaling downregulates Sox9 expression, thus supressing hair follicle SC specifi cation in other compartments than the bulge (Xu et al., 2015; Ouspenskaia et al., 2016). Th us, there are two signalling loops of Sox9 activation by Shh in the specifying SCs: Shh produced by Wnthi pocket cells directly activates Sox9 and also indirectly supports Sox9 expression by downregulating Wnt in the hair peg. At late embryogenesis, Sox9-expressing cells in the future bulge are also positive for the adult hair follicle SC markers Lhx2, Nfatc1 and Tcf3 (Nowak et al., 2008). Expression pattern of each of these transcription factors is initially broader than the exact pre-bulge region of the hair peg. Since future bulge residents are characterized by simultaneous expression of all these markers in a restricted, narrow zone, additional mechanisms likely exist for the sharp restriction of the specifying niche.

1.7 Mammary gland as an ectodermal appendage Th e other ectodermal organ whose development and homeostasis were studied in this work is the mammary gland. As a skin appendage, mammary gland development has much in common with the hair follicle. In mice, fi ve pairs of mammary placodes appear along precommitted areas on the fl anks around E11.5 (Propper et al., 2013; Cowin and Wysolmerski, 2010). Before puberty, the mammary gland grows relatively slowly. Th e placode gradually transforms into an ingrowth, a mammary bud, by E13.5. Th e mammary bud epithelium reorganizes into a light bulb-like structure while it plunges into condensed primary mammary mesenchyme. At E16 the mammary bud elongates and branches into the deeper lying tissue, secondary mammary mesenchyme, the precursor of the adult fatty stroma, the fat pad. Unlike the hair follicle, no specifi c dermal condensate can be detected underneath the mammary placode. Th e condensate becomes evident only at early bud stage (E12.5) coinciding with the onset of expression of certain mammary mesenchymal genes (Propper et al., 2013; Sakakura T., 1987). Between birth and puberty, the mammary gland continues isometric growth. During puberty, the mammary gland undergoes an explosive growth with extensive duct elongation and branching. As a response to ovarian stimulation, a new structure appears at the tips of the growing ducts – a terminal end bud, a point where from the duct bifurcates (Sternlicht et al, 2006). During pregnancy, the mammary gland undergoes further maturation to prepare for lactation. Th e tips of the branches transform into milk-producing alveoli. At this point the gland reaches its peak size. Limits for the growth is defi ned by the size of the fat pad, which provides a vital microenvironment for the growing mammary ducts (Sternlicht et al, 2006). Weaning initiates involution, another massive remodelling of the mammary epithelium. Th e obsolete milk-producing alveoli degrade, and the gland reverts close to a state similar to that prior to pregnancy. Th e rapid growth and involution upon pregnancy is tightly controlled by hormones and time-dependent orchestration of multiple signaling pathways. Th is mammary gland cycle of growth and regression resembles consecutive cycles of the hair follicle regeneration.

26 Review of the literature 1.8 Ectodermal dysplasias and hair disorders Ectodermal dysplasias (EDs) are genetic diseases characterized by missing or defects in two or more ectodermal appendages. In humans, nearly 200 diff erent conditions fi t the description of ED, and by 2008 a causative gene or at least an aff ected genome region were identifi ed for 67 clinically distinct EDs (Mikkola et al., 2009; Visioni et al., 2009; Itin, 2013). Similar development and utilization of similar genetic pathways of the ectodermal appendages explains the simultaneous defect of several ectodermal organs in EDs. Since Eda and Wnt pathways are the principal regulators of ectodermal appendage initiation and development, the majority of EDs are driven by mutations in these signaling pathways.

1.8.1 Monogenic hair disorders in humans Even though hair defects are typical for many EDs, disorders where hair follicle is the only ectodermal appendage aff ected are also common. By 2014 the causative gene in about 50 monogenic hair disorders had been identifi ed in humans (Duverger and Morasso, 2014). Th e conditions include disorders known as atrichia (hairlessness), hypotrichosis (sparse hair), hypertrichosis (excessive hair growth), as well as those where the hair structure is aff ected (Duverger and Morasso, 2014). Complete absence of hairs is called by term atrichia (from Greek). Total alopecia is another title for this condition, but it is worth to note that alopecia (“hair loss” in Latin) is a more general term referring to all hair disorders characterized by hair loss. So far, cases of total absence of hairs have been associated with the dysfunction of transcription factors. Mutation in one of such genes, HAIRLESS, causes several diff erent types of alopecia (Ahmad et al., 1998). FOXN1, a causative gene for the immunosuppressed Nude mouse phenotype, is mutated in patients with somewhat similar conditions, T-cell Immunodefi ciency and Congenital Alopecia (Frank et al., 1999). Hypotrichosis is a less severe type of hair loss characterised by normal to sparse hairs at birth, gradual hair loss during childhood and sparse hairs in adulthood. Th e most common causes of hypotrichosis are impaired hair development and regeneration, and defective anchoring of the hair shaft (Duverger and Morasso, 2014). Conditions of excessive hair growth or ectopic hair formations are generally named hypertrichosis. One interesting example is a small family of a rare disorder known as Congenital Generalized Hypertrichoses. No specifi c mutations have been related to this condition, but rearrangements and copy number variations have been implicated. For instance, a copy number variant upstream of SOX9 signifi cantly decreases SOX9 expression in the follicles of the patients with one of the conditions (Fantauzzo et al., 2012). Another case of hypertrichosis is caused by a mutation in FOXC2, leading to transformation of other ectodermal appendage, Meibomian gland, into eye lash, and growth of a second row of lashes (Fang et al., 2000). Another group of hair disorders aff ects hair structure. Mutations of keratins, the main structural component of the hair and the most abundant protein in the hair follicle cells, are the most frequent cause of thin, brittle and coarse hairs. Mutations in epithelial keratin (KRT) cluster on human chromosome 13, in KRT86, KRT81 or KRT83, lead to monilethrix, a condition with a bead-looking hair due to uneven thickness of the shaft . One commonly found condition associated with defects in hair curvature is “woolly” hairs. Th is phenotype is characterised by formation of fi ne and tightly curled hairs and is associated with mutations in several components

27 Review of the literature of tight junctions, , and others, as well as in the keratins of the IRS, KRT71 and KRT74 (McKoy et al., 2000; Shimomura et al., 2010; Fujimoto et al., 2012; Duverger and Morasso, 2014).

1.8.2 HypohidroƟ c ectodermal dysplasia Th e most common genetic disorder aff ecting several skin appendages in humans is a rare syndrome named Hypohidrotic Ectodermal Dysplasia (HED). HED is characterised by sparse hair, missing or abnormally shaped teeth, dry fragile skin due to disorders in functioning of sebaceous glands, and reduced sweating (Mikkola et al., 2009). In the fi rst years of life, patients are at risk for threatening hyperthermia and pulmonary infections (Clarke et al, 1987; Huttner, 2014). HED can be inherited in X-linked, autosomal recessive and autosomal dominant manners (Mikkola, 2009). HED patients carry mutations in the genes coding for the components of Eda signaling pathway. Th e most typical case of HED, X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) is caused by mutation in EDA and was the fi rst ED to be genetically characterised (Mikkola, 2009; Lefebvre and Mikkola, 2014). Female carriers of XLHED typically have a much milder clinical presentation than aff ected men (Huttner, 2014). Autosomal forms of HED are caused by mutation in EDAR or EDARADD and displays the same phenotype as XLHED. Much less common is a condition driven by mutations in the downstream eff ectors of the pathway, NFκB inhibitor IκBα and a component of NFκB complex IKKγ (Mikkola et al., 2009; Lefebvre and Mikkola, 2014). Mutations targeting the Wnt pathway, in Wnt ligand WNT10A gene, were reported as a cause of HED as well (Bohring et al., 2009), although the involvement of sweat glands is less clear.

1.8.3 Animal models in the study of ectodermal dysplasia and geneƟ c hair disorders ED and hair disorders are studied using several animal models including mice, rats, dogs, cattle and fi sh. Most of them carry XLHED, and the best characterised is the Tabby mouse which has a spontaneous mutation in the Eda gene (Falconer, 1952). Edar and Edaradd defi cient mouse strains display the same phenotype as Tabby, which resembles the human condition: defects in teeth, hairs, sweat glands, and other ectodermal derivatives (Sofaer, 1969; Headon et al., 2001; Yan et al., 2002). Two canine XLHED model resulted from an Eda splice site mutation associated with alternative splicing (Casal et al., 2005) and loss of 103 nucleotides from exon 2 in premature Eda transcript (Waluk et al., 2016). Dogs with XLHED have oligodontia, alopecia, and lack sweat and sebaceous glands (Lewis et al., 2010). Bovine XLHED was described in German Holstein cattle. Aff ected individuals were characterized by hypotrichosis, a reduced number of sweat glands, and tooth abnormalities such as missing incisors and missing or defective molars (Drögemuller at al., 2001). Fish models, medaka and zebrafi sh, with Eda or Edar defi ciency display nearly complete loss of scales, reduction in tooth structures and severe defects in fi ns (Iida et al., 2014; Harris et al., 2008). Research on mouse and dog XLHED models has given new hope for human patients. Recently there were no pharmacological therapies available to restore function in human disorders of development. Only the early recognition of the disease coupled with adequate

28 Review of the literature instructions for the parents could greatly reduce the risk of morbidity and mortality (caused by sweating defi ciency) during infancy (Bluschke et al., 2010). Antenatal and early neonatal Eda replacement with recombinant Eda protein in Tabby mice and XLHED aff ected dogs brought clinically signifi cant, sustained phenotype correction (Gaide and Schneider, 2003; Casal et al., 2007). Th is success provided the fi rst expectations for an eff ective, targeted therapy of XLHED in humans. Clinical trials of Fc-Eda from Edimer Pharmaceuticals were initiated in 2014-2015 in neonate male babies who were treated for the fi rst two weeks of life and will be followed until 10 years old (https://clinicaltrials.gov/ct2/show/record/NCT01992289). Taking into account the effi cacy of Eda therapy demonstrated on animal models, the only critical issue might still be the timing of drug administration and the dosage regime, as the developmental pace of ectodermal organs is diff erent between humans and animal models. In addition to the XLHED animal models, several other cases of hair abnormalities in animals have been described (Leeb et al., 2016). A hair disorder in cattle with typical X-linked inheritance is V- or Z-shaped hairless stripes noticible all over the body, so called lines of Blashko (Wollenberg and Eames, 2011). Th e condition was described for Nothern American Holstein female cattle, and discovered to be embryonically lethal in males (Eldridge and Atkeson, 1953). A causative gene for this phenotype was not characterized so far. In the time of this PhD work, two candidate genes for lines of Blashko condition in cows were identifi ed by our colleagues. One of the derivites of this PhD thesis work was characterization of the expression pattern of these genes in the hair follicle and confi rmation of their relevance as a causative genes for the hairless phenotype. Nowadays, genetic analyses have become easier in non-model animals and have gained increasing importance in identifying causative genes behind ectodermal dysplasia and/or hair disorders. Given the availability of numerous dog breeds, dog genetic testing is favourable to gain new knowledge on the genetic cause of a certain phenotype. For hair abnormalities in dogs, about 10 causative genes have been identifi ed. Th ese mutations aff ect hair pigmentation, hair length, or hair presence as part of ED syndrome (Leeb et al., 2016). Due to similar morphological characteristics of the human and canine hair follicles, the dog might be a promising model to study human hair cycle and hair follicle SC disorders (Welle and Weiner, 2016). Mutation of Fox transcription factor Foxi3 leads to canine ED in three hairless breeds: Mexican and Peruvian hairless dogs, and Chinese crested (Drögemuller et al., 2008). Individuals heterozygous for Foxi3 mutation have a sparse coat and aff ected dentition. Homozygote individuals are perinatally lethal. Chinese crested dogs that carry a Foxi3 mutation display variation in hairlessness from complete hair loss on the trunk to semi-coated, as well as variable tooth defects, but normal apocrine glands (Wiener et al., 2013). Th e Mexican hairless dog, Xoloitzcuintli, has been raised by Mexican Indians from Aztec era (1345-the early 16th century), and apparently have been used for curing rheumatism because of their high body temperature. In aff ected individuals, hairs are lost from most of the body accompanied by tooth defects. In the 80’s, one couple of Mexican dogs was introduced to Japan and mated with beagles to get a dog model for dermatological and dental research (Goto et al., 1986; Fukuta et al., 1991). Th e hairless beagles resulted from the cross showed that Foxi3 mutation manifested hair phenotype in multiple dog backgrounds.

29 Review of the literature 1.9 Forkhead box transcripƟ on factors Forkhead DBD folds into a variant of the helix-turn-helix motif and is made up of three helices and two characteristic large loops, or “wings.” Th erefore, it is also named the “winged- helix” DBD (Kaestner et al., 2000). Th e fi rst forkhead gene was described in Drosophila melanogaster (Weigel et al., 1989). Comparative genome analyses have shown that the number of Fox transcription factors appears to have increased during evolution, with a greater number identifi ed in vertebrates than in invertebrates (Lehmann et al., 2003). Among the organisms for which genome sequences are known, there is a correlation between anatomical complexity and Fox gene number: 4 in Saccharomyces cerevisiae, 15 in Caenorhabditis elegans, 20 in Drosophila melanogaster, and 39 in humans (Carlsson et al., 2002). Th ese data suggest that the origin and expansion of Fox genes is positively correlated with eukaryotic complexity. Th e mammalian Fox transcription factor superfamily contains more than 40 genes in 19 subfamilies, from Foxa to Foxs. Th e Fox factors play myriad roles in gene regulation and act in all contexts of homeostasis of the organism. Fox family members function as transcription factors in the classic sense by recruiting chromatin remodelling factors, as well as pioneering factors by opening condensed chromatin to initiate regulatory control of gene transcription (Kaestner, 2010, Lalmansingh et al., 2012). FoxP transcription factors are perhaps the best-known members of the Fox family due to the famous FoxP2 “speech” gene (French and Fisher, 2014). Subfamilies of FoxA and FoxO are also well-characterized (Lalmansingh et al., 2012). Th e memebers of FoxA subfamily function as pioneer transcription factors to initiate transcriptional regulation. Classic transcription factors depend on chromatin modifying enzymes to alter the local chromatin structure, however, FoxAs open up chromatin directly. FoxAs interact with specifi c DNA sequences within compacted chromatin and promote nucleosome de-compaction, thereby making the genomic region more amenable to regulatory factor binding and transcriptional control (Kaestner, 2010), “pioneering” access to DNA. Pioneer factors Foxa1 and Foxa2 bind to target sites in chromatin at early developmental stages, remain bound to DNA during division, and remodel chromatin to prepare for the activation of lineage-specifi c genes later in development (Zaret and Carrol, 2011). FoxP proteins are examples of traditional transcription factors which possess distinct transcriptional regulatory and DBD. Th e N-terminal half of FoxPs contains a transcriptional repressor region with a zinc-fi nger/ motif suggesting their role in gene repression. Dimerisation with another FoxP protein favours DBD stabilisation, as well as interaction with other transcriptions factors: Nfatc, NFκB and Runx1 (Loisou et al., 2011; Ono et al., 2007). FoxO sub-family is an example of proteins that displays both pioneering and classic transcriptional functions. Th is dual ability places FoxOs in a novel and unique class that combines chromatin remodelling and transcriptional regulatory activity in a single protein (Lalmansingh et al., 2012).

30 Review of the literature

A subset of Fox proteins interacts with highly condensed mitotic chromatin implicating a ‘bookmarking’ function to maintain specifi c genomic regions in a transcriptionally competent state during cell cycle progression (Cirillo and Barton, 2008). Transcription factors in general are displaced from the tightly organized mitotic chromatin and resume occupancy following mitosis. However, some factors, including certain Fox proteins remain stably associated with condensed mitotic chromatin. For instance, FoxM1 is a master regulator of cell cycle genes; its expression is required for proper chromosome segregation and mitotic progression (Priller et al., 2011). Th e association of Fox proteins with condensed mitotic chromatin is not surprising given that pioneering function involves interactions with compacted chromatin. Continuous mitotic chromosome binding by Fox proteins may serve to maintain key target genes in a “transcriptionally competent” state for rapid activation or repression following mitotic exit. Th is “bookmarking” eff ect may thus be an adaptive mechanism for transmitting epigenetic information to daughter cells (Arora et al., 2012; Lam et al., 2013).

1.9.1 Fox transcripƟ on factors in the hair follicle Fox proteins with a defi ned role in hair follicle homeostasis include Foxn1, Foxc1, and Foxi3 (see Chapter 1.9.2). Mouse KO for Foxn1 are the Nude mice which are widely used for transplantation assays because of their supressed immune system and lack of graft rejection. Foxn1-defi cient mice are bald since hair shaft formation is severely aff ected (Mecklenburg et al., 2004). Recently the role of another Fox transcription factor was uncovered in two independent studies (Lay et al., 2016; Wang et al., 2016) (Table 2). Foxc1 was shown to be a key gene in regulation of hair follicle SC quiescence. Th e loss of Foxc1 leads to “one bulge” phenotype, also typical for Lhx2 cKO and caused by impaired hair anchoring. However, the initial reason behind the phenotype diff ers between Lhx2 and Foxc1 mutants, since Foxc1 deletion resulted in activated SC proliferation and consequent E-cadherin downregulation (Folgueras et al., 2014; Lay et al., 2016).

1.9.2 Foxi3 Th e Foxi family consists of three members in vertebrates: Foxi1, Foxi2, and Foxi3. Murine Foxi3 contains 4533 nucleotides across one intron and two exons, splicing of which results in a 1278 bp transcript that codes for the 425 amino acid protein (MGI:3511278; NCBI Gene:232077). Foxi3 is expressed early in development in sensory placodes and pharyngeal arches of mouse and chick embryos and is required for craniofacial patterning and ear formation (Edlund et al., 2014; Khatri and Groves, 2013). Zebrafi sh Foxi1 gene is expressed in a similar pattern to mammalian Foxi3 in the pharyngeal epithelium during arch development. A zebrafi sh Foxi1 mutant, hearsay, lacks an otic vesicle and presents severe defects in skeletal structures that develop from the anterior arches (Nissen et al., 2003; Solomon et al., 2003; Hans et al., 2007).

31 Review of the literature

Studies of mouse Foxi3 mutants have demonstrated that Foxi3 regulates otic structure development through cross-talk with Fgfs. Foxi3 activates Fgf8 expression and thus provides survival cues for neural crest cells populating the pharyngeal arches (Edlund et al., 2014). Th e link between Foxi3 and Fgf signalling was confi rmed in the study of chick otic placode development (Khatri et al., 2014). Even though Foxi3 is expressed very early in the otic preplacodal area, initiation of the fi rst events of otic placode development was not aff ected in Foxi3 morpholino knockdown. Foxi3 was necessary for otic placode induction only later, at a time aft er Fgf signal transmission, suggesting that Foxi3 was required to confer competence for otic induction in response to Fgfs. Early Foxi3 expression but late requirement allow speculating that Foxi3 could act as a pioneer factor in otic placode induction occupying certain otic enhancers and enabling Fgfs to activate the diff erentiation program (Khatri et al., 2014). Th is hypothesis is supported by the data that zebrafi sh Foxi1 remains bound to the condensed chromatin during mitosis and creates stable DNAseI sensitive changes in the chromatin (Yan et al., 2006). Severe phenotypes of the dogs with Foxi3 mutations suggest that Foxi3 is crucial for the formation of ectodermal appendages. However, prior to this study, there were no detailed reports on Foxi3 expression pattern and nothing was known about its transcriptional regulation in ectodermal organs. In this work, the role of Foxi3 in the hierarchy of the key signalling pathways guiding hair follicle induction, growth and renewal was explored.

32 Aims of the study 2 AIMS OF THE STUDY

Identifi cation of the causative mutation of hairless phenotypes helps to elucidate biological function of the genes. Mutation of Foxi3 drives hair and tooth disorders in several dog breeds suggesting a potent role in regulation of hair follicle development or maintenance. Th is work focused on characterization of the function of Foxi3 in hair follicle homeostasis and the mechanisms of its action. Hair disorders are oft en coupled with defects in other ectodermal organs. Th e impact of Foxi3 was also studied in another ectodermal appendage, mammary gland. Identifi cation of the causative gene for a bovine hair disorder and analysis of its expression pattern in murine hair follicles was another objective of the work.

Aims of the thesis were:

I To study Foxi3 expression during hair follicle development and regeneration.

II To identify upstream regulators of Foxi3 and its downstream targets.

III To characterize the consequences of Foxi3 loss in the hair follicle and mammary gland using Foxi3 de¿ cient mouse models.

IV To study expression pattern of Tsr2 and Erccl6l, a causative genes for hair disorders in cows.

33 Materials and methods 3 MATERIALS AND METHODS

Mouse strain Reference Used in publication Bat-Gal Maretto et al., 2003 II C57Bl/6 Harlan I, II, III Eda-/- Harlan I Foxi3-/- Kindly provided by Andrew Groves II Foxi3fl oxed/fl oxed Kindly provided by Andrew Groves II Hsd:ICR(CD-1) Jackson Laboratory II K14-Cre Huelsken et al., 2001 II K14-Cre43 Andl et al., 2002 II K14-CreERT Jackson laboratory, stock no. 005107 II K14-Eda Mustonen et al., 2003 I Lgr5CreIRES-GFP-ERT2 Jackson laboratory, stock no. 008875 II NF-κB gal Bhakar et al., 2002 II NMRI Jackson Laboratory I, II, III Nude Harlan II Shh::GFPCre Harfe et l., 2004 Unpublished data Sostdc1-/- Kassai et al., 2005 I

Following primary antibodies were used for immunostaining data for the publication II.

Antibodies Manufacturer AE13 (K40) LS Bio LS-C193780 AE15 (Trichohyalin) LS Bio LS-C82335 BrdU Th ermo Fisher Scientifi c, MS-1058-P1 Caspase3 Cell Signalling Technologies, D175, lot#9961S Foxi3 Santa Cruz, L-17, SC-324865, lot# KO112 Gata3 Life Span Biosciences LS-B4879 lot# 56082 Keratin 15 AbCam, ab52816, cat# 1954-1, EPR1614Y Lef-1 Cell Signaling Technology Lhx2 Santa Cruz, C-20, SC-19344, lot# J1514 Nfatc1 Santa Cruz, 7A6, SC-7294 lot# B1214 P-cadherin R&D Systems, AF761, lot# FHQ02 Sox9 Millipore, AB5535, lot# 2167153

34 Materials and methods

Methods and techniques are described in details in the original publications of these thesis.

Method Used in publication Basic laboratory techniques Histology I, II, III Carmine alum staining of mammary glands Unpublished data Microscopy I, II, III Techniques with the use of tissue culturing Induction of gene expression with protein soaked bead I Induction of gene expression with protein-enriched medium in hanging drops I Phenotype rescue experiments with cultured back skin explants II Gene expression analysis Whole mount in situ hybridization I, II, III Radioactive in situ hybridization I, II, III Probe cloning for in situ hybridization III Quantitative real-time PCR I, II Aff ymetrix Mouse Exon 1.0 ST Microarray analysis II X-gal staining of LacZ reporter tissue samples II Detection of proteins Immunohistochemistry (cell proliferation assay – BrdU staining) II Immunofl uorescence II Mouse work Skin graft ing to the dorsum of Nude mice II

In situ hybridization probes Reference Used in publication Dkk4 Fliniaux et al., 2008 II Erccl6l Publication III III Foxi1 Ohyama and Groves, 2004 II Foxi2 Ohyama and Groves, 2004 II Foxi3 Ohyama and Groves, 2004 I, II Lgr6 Generated by Emma Juuri, Th esleff group Unpublished data PTHrP Voutilainen et al., 2012 Unpublished data Runx1 Yamashiro et al., 2002 Unpublished data Shh Vaahtokari et al., 1996 II Tsr2 Publication III III

35 Results and discussion 4 RESULTS AND DISCUSSION 4.1 Expression paƩ ern of Foxi3 in ectodermal appendages (I and II) Given that Foxi3 is expressed in primary hair placodes and in developing teeth (Drögemuller et al., 2008; Jussila et al., 2015), we tested whether Foxi3 was present in other ectodermal organs. Expression of Foxi3 was studied in the mammary gland and salivary glands. Whole mount in situ hybridization (ISH) revealed Foxi3 expression in the third and forth mammary gland pairs. Using more sensitive radioactive in situ hybridization (ISH), Foxi3 was detected in all fi ve pairs of the mammary glands at E12.5. Foxi3 expression waned soon aft er and was not detected in the mammary buds at E14.5. Th is suggests that Foxi3 could be required for the early stages of mammary gland development, but is dispensable at the later time points. Foxi3 expression was detected in two other exocrine glands studied: the salivary gland and in the eccrine sweat gland on the paw. As previously described (Ohyama and Groves, 2004), Foxi3 was expressed in the branchial pouches. In addition, Foxi3 expression was found in the nails, around the anal opening, in the cranial placode-derived otic vesicle and optic cap.

4.1.1 Foxi3 displays dynamic expression in developing and cycling hair follicles (I and II) To deepen previous knowledge on Foxi3 expression (Drögemüller et al., 2008), we identifi ed localization of both Foxi3 mRNA and protein at all stages of hair follicle morphogenesis and cycle. Using ISH we showed that Foxi3 mRNA was expressed in the epithelium of the placodes of all hair follicle types. Expression was maintained in the epithelium also during the downgrowth of the hair follicle. Th e same results were obtained in immunostaining with Foxi3 antibody. Specifi city of the antibody was confi rmed by negative staining of Foxi3 null embryos. In more advanced hair follicles, Foxi3 mRNA was no longer detectable. However, by immunostaining we identifi ed Foxi3 in a subset of cells in the center of the follicle above the Mx. In fully formed follicles, Foxi3 was no longer detectable. We followed the localisation of both Foxi3 mRNA and protein during the fi rst hair cycle. Foxi3 reappeared at catagen in the cells of the epithelial strand and became restricted to the hair germ at telogen. At the onset of anagen, Foxi3 expression expanded into TA cells. Similarly to morphogenesis, at the time of shaft generation, Foxi3 disappeared from all cell populations of the anagen follicle. Th is dynamic expression suggests that Foxi3 might play distinct roles in gene regulation at diff erent stages of development in many cell types.

4.1.2 MulƟ ple pathways regulate Foxi3 expression (I and unpublished) To gain insights into possible pathways regulating Foxi3, its expression was studied in mouse strains with perturbed developmental signaling pathways. Since Foxi3-defi cient dogs exhibit an ED condition, which is driven by Eda-mutation in many animal species, the fi rst hint was to analyze Foxi3 expression in Eda-null Tabby (Pispa et al., 1999) and Eda-overexpressing K14-Eda (Mustonen et al., 2003) mouse strains. Using whole mount ISH, an increase in Foxi3 expression

36 Results and discussion was detected the hair placodes and mammary buds of K14-Eda mice. Foxi3 was also detected in the supernumerary mammary buds that are characteristic of K14-Eda mice. Foxi3 was not found in mammary buds of Eda-defi cient mice. Similarly, Eda enhanced Foxi3 expression in tooth epithelium of K14-Eda mice at e13.5, prior to tooth bud stage. Th is suggested that Foxi3 might be a downstream target of Eda in diff erent ectodermal appendages. To test this possibility further, we studied Foxi3 induction by a short-term treatment with recombinant Eda protein. For that, E14.5 back skins were incubated in hanging drops of Eda-enriched medium. Foxi3 expression was analysed by quantitative real time PCR (qRT-PCR) and showed 12-times increase aft er 2 hours of treatment and more than 20-times increase aft er 4 hours, compared to control samples. Since the activation response was so rapid, this data suggested that Foxi3 is a direct Eda target in the developing hair follicles. In support of our conclusion, a recent study on Eda/NFκB target genes identifi ed Foxi3 among the most downregulated genes in NFκB-defi cient (ΔN) mice (Tomann et al., 2016). Several other proteins with a known impact on ectodermal organ development were analysed in an in vitro experimental assay for their eff ect on Foxi3. Beads soaked in Shh, Fgf4 and 10, BMP4 and Activin A were placed on E13.5 back skins, cultured overnight and the expression of Foxi3 was analyzed by whole mount ISH. Activin A was the only protein that caused a defi nite eff ect on Foxi3 expression. Th is result was confi rmed by hanging drop culture method in which wild type skin was treated with Activin A enriched-medium, that drove a modest, but statistically signifi cant 4-times activation of Foxi3 aft er 4 hours of treatment. Activin has been shown to upregulate the expression of Edar (Laurikkala et al., 2001; Laurikkala et al., 2002). However, Edar gene expression was not induced in the same is experiment, suggesting that Activin may directly regulate Foxi3 expression. It was shown that in the hair follicle SCs Activin/Tgfβ pathway lies downstream of Sox9 (Kadaja et al., 2014). It might be that in the developing hair follicle Foxi3 is regulated by Sox9/Activin/Tgf signalling axis. To learn more about Foxi3 regulation in in vivo, Foxi3 expression was analyzed in mice defi cient for Sostdc1, Follistatin (I) and Shh (unpublished, Fig. 6) all of which showed normal Foxi3 expression.

Control Shh KO

Foxi3

Figure 6. Foxi3 expression was not aff ected in Shh KO embryos. Foxi3 expression was analyzed by whole mount ISH at E14.5.

37 Results and discussion 4.2 Impact of Foxi3 deleƟ on on ectodermal appendage development (II and unpublished) For the functional studies of Foxi3, we used germline-deleted Foxi3 null mice (hereaft er Foxi3 KO) (Edlund et al., 2014). Foxi3 KO were created in the laboratory of Andrew Groves and kindly provided to us. Foxi3 KO mice are perinatally lethal due to severe craniofacial abnormalities associated with early Foxi3 deletion from the branchial arches, leading to absence of the lower jaw and parts of the upper jaw. Hence, only embryonic ectodermal organs and transplanted tissues of Foxi3 KO mice could be characterized, and analysis of teeth was heavily compromised due to head malformation. To elucidate the impact of Foxi3 on postembryonic ectodermal organ development, we used conditional Foxi3 KO mice with epithelium-specifi c ablation of Foxi3 by K14-Cre. Foxi3 locus was modifi ed by the insertion of two loxP sites upstream and downsream of exon 2 that enabled to excise the fl anked (fl oxed) gene segment through Cre-mediated recombination. To generate conditional Foxi3 KO, Foxi3fl oxed/fl oxed mice were crossed with Foxi3del/wt; K14-Cre+/-. Foxi3del/fl oxed; K14-Cre+/- (hereaft er referred to as Foxi3 cKO) were obtained and exhibited a mutant phenotype.

4.2.1 Hair follicle downgrowth and hair cycling were aī ected in the absence of Foxi3 (II) Unlike Foxi3-defi cient dogs with an ED condition, heterozygous Foxi3 mice were normal. Th e mouse model was generated by deletion of exon 2 of the Foxi3 gene, while the hairless phenotype in dogs is caused by a 7 bp duplication in exon 1 which produces a frameshift and a premature stop codon. Although it is unclear whether the mutant protein is actually expressed, with more than 95% of the normal Foxi3 protein missing, it is unlikely that the mutant protein is functional. Any mutant protein produced will contain 218 amino acids and could theoretically interfere with normal cellular function in a dominant-negative mechanism (Drögemüller et al., 2008). However, why the deleterious eff ect of Foxi3 loss is evident in hair follicles and teeth, but not in other tissues where Foxi3 is expressed, is currently unknown. Certain compensatory mechanisms in gene regulation might exist in some tissues and compensate for the mutant phenotype. Homozygous Foxi3 KO embryos displayed a hair phenotype. Even though hair follicle initiation was not aff ected in Foxi3 KO and placodes arose on time at E14.5, a day later, impaired invagination of the hair germ was evident. Th e downgrowth phenotype persisted throughout embryogenesis, and prior to birth, Foxi3 KO embryos had fewer and shortened hair follicles compared to control littermates. Cellular proliferation was decreased in Foxi3 KO skin at E15.5, but apoptosis was not increased. Graft ing of back skins from E18.5 embryos to Nude mice allowed following postnatal fate of Foxi3 KO hair follicles. Aft er three weeks, control transplants gave rise to bundles of hairs, while Foxi3 KO graft s gave rise to very few hairs, indicating that the rest of the follicles degraded. Th is suggests that Foxi3 is required for proper downgrowth of the hair follicles during embryogenesis and partially for postnatal hair formation as well. Th ose few hair follicles that proceeded to grow had normal morphology without gross defects, suggesting that under some conditions, Foxi3 is dispensable for hair growth. Skin graft s of Foxi3 KO skin resembled the hairless condition in Foxi3 null dogs.

38 Results and discussion

Overall, mouse embryonic Foxi3 KO hair phenotype was notably milder compared to Foxi3 mutant dogs. Curiously, the phenotype was present only in the inbred C57Bl/6 strain. Foxi3 KO in the outbred ICR background did not show any obvious embryonic hair phenotype. Of note, Foxi3 mutation in dog manifests the phenotype in at least three dog breeds, including the beagle background (Drögemüller et al., 2008; Fukuta et al., 1991). To search for additional mechanisms which might compensate for Foxi3 loss in hair development, we addressed the possibility of redundancy of Foxi genes. We studied expression of Foxi1 and Foxi2 in developing hair follicles in wild type and Foxi3 KO embryos. Even though a previous study suggested that Foxi2 is expressed in ectodermally derived organs: kidney, thymus, certain areas of the brain and in some other organs (Wijchers et al., 2005), we could detect neither Foxi2 nor Foxi1 in the hair placodes of wild type embryos. Foxi genes were not activated in Foxi3 KO either. Th e same result was obtained in developing teeth (Jussila et al., 2015). Absence of phenotype in homozygous mice indicates existence of compensatory mechanisms, but this is not likely to be redundancy with other Foxi genes. We have examined the activity of the main signaling pathways that guide ectodermal organ development: Wnt/β-catenin, Eda/NFκB and Shh, in Foxi3 KO hair follicles. We studied the activity of Wnt pathway using Bat-Gal reporter mice (Maretto et al., 2003) and NFkB activity using NFkB-reporter mice (Bhakar et al., 2002), and did not observe any apparent diff erences in Wnt and NFkB signaling in hair follicles of Foxi3 KO embryos. Likewise, expression of Wnt coreceptor Lgr6 was not downregulated in Foxi3 KO (Fig. 7), and Lef-1 activity was not aff ected as well.

Control Foxi3 KO E19.5

Lgr6

Figure 7. Wnt signaling was not aff ected during hair follicle morphogenesis in Foxi3 KO. Expression of Lgr6 was similar in control and Foxi3 KO embryonic hair follicles. Lgr6 expression was detected by radioactive ISH at E19.5. Upper row – bright fi eld image; lower row – dark fi eld image showing radioactive ISH signal. Scale bar=50 μm.

39 Results and discussion

Given that Shh pathway is a known regulator of hair follicle downgrowth (St-Jacques et al., 1998, Chiang et al., 1999; Mill et al., 2003), we analysed Shh expression in Foxi3 KO. We did not detect a change in Shh mRNA level in Foxi3 KO at placode stage. Previously, it was discussed that Foxi3 expression is not aff ected in Shh KO embryos, suggesting that in embryonic hair development, Shh and Foxi3 signaling are uncoupled. Th e downgrowth phenotype of Foxi3 KO indicates that there is another signaling axis, in addition to the Shh pathway, regulating follicular growth. Next we tried to rescue the downgrowth defect in Foxi3 KO skin explants by culturing them in the presence of signalling molecules with a known function in hair follicle induction and growth, such as Wnt3a and Fgfs. Aft er 4 days in culture in a control medium, hairs on the control skin explants grew signifi cantly, while Foxi3 KO hair follicles persisted as placodes during the fi rst days in vitro, and degraded later. Th e in vivo Foxi3 KO hair phenotype is less dramatic: hair follicles grow slower, but do not degrade at least until perinatal death of the embryo. Th is signifi es that in in vitro conditions, Foxi3 functions as a survival factor for the developing hair placodes. As previously described, in the graft ing experiment we observed similar results. Treatment of Foxi3KO skin explants with Wnt3A/R-spondin2 or Fgf10 did not rescue hair follicle development. A previous study had shown that Foxi3 regulates Fgf8 expression in pharyngeal arch development in mouse embryo (Edlund et al., 2014). We tested Fgf8-enriched medium, but did not observe any promoting eff ect for hair follicle survival. Apparently, one factor is not enough to compensate the deleterious eff ect of Foxi3 ablation. Th e impact of Foxi3 ablation on postnatal ectodermal organ development was studied using Foxi3 cKO. Two diff erent K14-Cre lines, a knock-in (Huelsken et al., 2001) and a transgenic (Andl et al., 2004), were used to generate Foxi3 cKO. Conditional mutants resulted from both crossings and displayed similar hair phenotypes. In contrast, the teeth were more severely aff ected in mutants generated with the transgenic K14-Cre, which is activated earlier during development before tooth initiation. Th erefore, all subsequent analyses of the Foxi3 cKO phenotype was completed on conditional mutants from the crossings with the transgenic K14-Cre line. Foxi3 cKO were born in a Mendelian ratio, were viable, but consistently smaller than control littermates (Jussila et al., 2015). Foxi3 cKO mice had sparse coat hair, visible from the time when hairs appear. Th e phenotype was stable throughout life and even progressive in some individuals, which gradually lost hairs with age. Even though Foxi3 cKO displayed a hair phenotype, it was milder than in the Foxi3 mutant dogs with ED. Effi ciency of the K14 driver was high and unlikely accounts for the mild phenotype, as we never observed residual Foxi3 expression in Foxi3 cKO. Apparently, as in case with ICR Foxi3 KO, mixed background of our Foxi3 cKO (NMRI::ICR) might explain the less severe phenotype in the murine coat compared to dogs. To determine the role of Foxi3 in regulation of hair regeneration and maintenance, we characterised the progression of Foxi3 cKO follicles through the fi rst hair cycle. By late morphogenesis at P10, Foxi3 cKO follicles displayed morphological abnormalities, formed cysts fi lled with fragmented hair shaft material. Foxi3 cKO hair follicles delayed in proceeding through catagen and telogen, many hair follicles had enlarged sebaceous glands and with time transformed into a glandular compartment lacking follicle characteristics. Th is indicates that Foxi3 is not required to generate proper hair shaft morphology, but rather for hair follicle morphogenesis and cycling.

40 Results and discussion 4.2.2 Foxi3 is dispensable for mammary gland development (unpublished) Expression of Foxi3 in early mammary bud prompted us to study mammary gland development and function in Foxi3 defi cient animals. First we explored whether Foxi3 could play a role in mammary gland induction and early development. Foxi3 null embryos had normally patterned mammary buds at E12.5, which developed into mammary gland trees by E18 (Fig. 8, A, B). Formation of Eda-induced supernumerary mammary primordia in K14-Eda::Foxi3 KO embryos was not aff ected by loss of Foxi3 (Voutilainen et al., 2015). Pubertal mammary gland development in Foxi3 cKO females was normal as well. Th e size of the gland and its branching analysed at 6 and 11 weeks old were similar in Foxi3 cKO and control sisters (Fig. 8, C-F). Mutant females were able to feed their pups as successfully as control littermates as determined by pup weight. In essence, these data suggest that despite Foxi3 expression at early stages of development, the mammary gland was unsusceptible to Foxi3 ablation.

Control Foxi3 KO AB

E18.5 Control Foxi3 cKO CD

LN LN

6 weeks old EF

LN LN

11 weeks old

Figure 8. Mammary gland development was not aff ected by the loss of Foxi3. (A, B): Normally formed ductal tree of the third pair of mammary glands in Foxi3 KO (B) embryos compared to control (A) at E18.5. (C-F): Postnatal mammary gland branching in Foxi3 cKO (D, F) was similar to control sister (C, E) at the stages indicated, forth pair of mammary gland. Mammary epithelium of was visualized by carmine alum staining. LN – lymph node.

41 Results and discussion 4.3 Genes and pathways downstream of Foxi3 (II and unpublished) 4.3.1 A number of hair follicle stem cell speciĮ c genes are downstream of Foxi3 (II and unpublished) To identify the molecular mechanisms behind the Foxi3 hair phenotype, we analysed diff erences in gene expression in skin epithelia of Foxi3 KO and wild type littermates by microarray on Aff ymetrix Exon 1.0 ST. For that we chose the E15.5 stage when the fi rst apparent phenotype was noticed. Around 500 genes were diff erentially expressed in Foxi3 KO, the majority were downregulated, indicating that in hair development, Foxi3 is mainly a transcriptional activator. Among the short list of the genes interesting in terms of hair follicle biology, very few genes were associated with hair follicle initiation or embryonic development. Many more putative target genes represented well-known hair follicle SC markers: Lhx2 (Rhee et al.; 2006), Runx1 (Osorio et al., 2008), (Li et al., 2012), Hmga2 (Chen et al., 2012), Nft ac1 (Horsley et al., 2008), and the genes with a described postnatal hair cycling phenotype, for instance, Hairless (Hr) (Kazantseva et al., 2006). We validated the microarray data with qRT-PCR on independent pairs of E15.5 Foxi3 KO and wild type samples. Two genes which are expressed from the initiation stage, Dkk4 and PthrP, were up- and down-regulated, respectively, in Foxi3 KO epithelium. Dkk4 is an inhibitor of the Wnt receptor, and its upregulation in Foxi3 defi cient hair follicle might point to a link between Foxi3 and Wnt pathway, which we could not, however, detect in our previous experiments. We showed a statistically signifi cant downregulation of Lhx2, Nfatc1, Runx1 and LipH genes in Foxi3 KO. Expression of some of these genes was studied further by radioactive ISH. PTHrP and Runx1 were downregulated in developing Foxi3 KO hair follicles (Fig. 9).

Control Foxi3 KO E18.5

PTHrP

Runx1 Figure 9. Certain genes responsible for stem cell specifi cation and hair follicle development were downregulated in the absence of Foxi3. Radioactive ISH revealed downregulation of Runx1 and PTHrP in Foxi3 KO hair follicles at E18.5. Scale bar=50 μm.

42 Results and discussion

Parallel to the microarray performed on E15.5 Foxi3 KO cutaneous epithelia, a PhD student from our closest collaborating Th esleff group, Maria Jussila, performed a microarray was carried out on E13.5 Foxi3 cKO molar epithelium (Jussila et al., 2015). Very few genes targeted by loss of Foxi3 were overlapping between skin and molar epithelia. Th e reason could be that SC-associated genes might not be diff erentially expressed in molar epithelium as mouse molars do not grow continuously and there is no SC niche. In contrast, mouse incisor has a specifi c compartment housing SCs, the cervical loop (Th esleff and Tummers, 2009). Genome profi ling of Foxi3-defi cient cervical loops will be needed to reveal whether tooth SC-signature genes are regulated by Foxi3 and if there are similarities with Foxi3 KO skin epithelial gene profi ling. In molar morphogenesis, Foxi3 inhibits dental diff erentiation and prevents formation of the tooth signaling centers, the primary and secondary enamel knots (Jussila et al., 2015). Unlike in skin epithelium, among more than 700 diff erentially expressed genes in molar, 431 were upregulated, including components of the Fgf, Shh, Wnt and Bmp pathways. Th is means that loss of Foxi3 promotes diff erentiation of the dental epithelium toward the enamel knot. Ultimately, these results indicate that Foxi3 in the dental epithelium acts as an inhibitor of diff erentiation that prevents the epithelium from precociously acquiring the fates of cervical loops and enamel knots and probably also of ameloblasts (Jussila et al., 2015). Th is parallels Foxi3 function in the hair follicle: the undiff erentiated and proliferative state of the dental epithelium resembles features of the hair germ SCs. Th e hair follicle microarray was performed on isolated back skin epithelia, not on sorted placodal cells. Th is results in dilution of any placode-specifi c signal by the data from interfollicular epidermis, where there should not be any Foxi3-driven changes in gene expression. Th is specifi cally accounts for those genes that are ubiquitously expressed in the epidermis, with relative up- or down-regulation in the placode. Subtle diff erences in developmental stage or individual sample diff erences might bring fl uctuation in gene expression signal, therefore raising the threshold for signifi cant changes in expression. Th is might result in masking of some Foxi3 targets, and thus they would not be included in the list of diff erentially expressed genes. Th is also could be a reason of a moderate fold change of expression in our microarray data. Th ere is no perfect marker for placodal cells that would allow their sorting. P-cadherin used for this purpose is expressed in the interfollicular epithelium at a low level (Sennett et al., 2015). Recently, in P-cadherin-based sorting of embryonic skin cell populations, Foxi3 was used as a placodal-signature to validate the identity of the sorted cells (Sennett et al., 2015). Nevertheless, the validation of Foxi3 target genes described earlier confi rmed that the chosen approach for our microarray was successful.

4.3.2 Foxi3 regulates hair follicle stem cell speciĮ caƟ on during morphogenesis We analysed expression of hair follicle SC markers in Foxi3+ cells. Using double immunostaining we showed that Foxi3-expressing cells mark the same population as Lhx2+ cells. Foxi3 appears in these cells earlier, by E14.5, when no Lhx2 signal is detected yet. A population of Foxi3+ cells express also NFATc1. We have studied an early marker of hair follicle SC Sox9, even though it was not detected in the microarray. In the hair placode and hair peg, Foxi3+ and Sox9+ cells represent distinct populations except for a small number of double positive cells at the middle of the growing hair peg, between Foxi3+ lower part and Sox9 + upper part. At more advanced

43 Results and discussion stages of development, Foxi3+ cells in the center of the follicle are positive for Sox9, however, Foxi3 is absent from Sox9+ ORS cells, the precursors of the bulge. In the telogen hair follicle Foxi3 marked the population of activated SCs in the hair germ and was coexpressed with Lhx2 and Sox9. Th us, Foxi3 is expressed in the hair placode earlier than hair follicle SC markers Lhx2 and Nfatc1. In the hair peg, the upper cell layers are Sox9+, while the lower cells activate Lhx2. Foxi3+ cells occupy middle-lower area of the placode, they express a low level of Sox9 at the middle part and are Lhx2+ in the lower part. As the follicle develops, areas of Sox9 and Lhx2 expression are partly mixed (Nowak et al., 2008). Foxi3 is down-regulated in Lhx2 and Sox9 double positive cells and is activated in the band of the middle cells, which also express Sox9, but are negative for Lhx2. To follow SC niche specifi cation in the absence of Foxi3, we analyzed the expression of SC signature genes in Foxi3 KO. We did not detect Lhx2 in primary hair placodes of Foxi3 KO embryos, probably refl ecting the downgrowth defect fi rst noticeable at this stage. Similarly to Lhx2, Nfatc1 was not detected in Foxi3 KO at the same stage when it appeared in control littermates. Th is indicates that the initial specifi cation of hair follicle SC precursors was aff ected in the absence of Foxi3. Recently the impact of Lhx2 in downgrowth of hair placodes was identifi ed and linked to disrupted Eda pathway in NFκB ΔN mice (Tomann et al., 2016). Microarray and ChIP on E14.5 placodal cells sorted with NFκB-EGFP from ΔN embryos indicated that Lhx2 is a direct downstream target of Eda/NFκB and regulates hair follicle downgrowth through the TGFβ pathway. Similarly, Lhx2 was decreased in the microarray from Foxi3 KO epithelia and in immunostaining of Foxi3 KO embryos, however NFκB signaling was not changed in the absence of Foxi3. In contrast, Foxi3 was downregulated in NFκB ΔN mice (Tomann et al., 2016). Th is might indicate that, in addition to NFκB, Foxi3 takes part in Lhx2 activation and simultaneous binding of NFκB and Foxi3 to the Lhx2 promoter drives its expression. Signaling through Foxi3, NFκB and Lhx2 might regulate hair placode downgrowth beyond initiation while at the later stages Shh functions for the downgrowth. Next we analysed expression of SC markers in Foxi3 KO at late embryogenesis, when the SC niche was forming. Foxi3 KO hair follicles showed normal expression patterns for Lhx2 and Sox9. In contrast, Nfatc1 was dramatically downregulated in Foxi3 KO. Another SC marker, K15, was also noticeably downregulated in Foxi3 KO hair follicles but not in the epidermis where it is expressed at high levels during embryogenesis. Downregulation of structural protein K15 and transcription factor Nfatc1 indicates a change to keratinocyte fate in the absence of Foxi3. Lineage tracing experiments have shown that Nfatc1+ cells appear at the hair peg stage, do not contribute to follicle downgrowth, but make up most of the adult hair follicle SCs (Xu et al., 2015). Laser ablation of Nfatc1+ cells did not have an impact on bulge formation. Th ese cells were completely substituted and the new SCs were able to express Nfatc1 and initiate fi rst anagen (Xu et al., 2015). Th is underscores the plasticity between follicle cell populations and shows that location of the SC niche, rather than marker expression, determines cell diff erentiation potential. However, the exact SC potential of these new-born SCs was not followed in multiple postnatal cycles. Th e source of these cells is not known, nor when they restore SC marker expression. Th e most probable cell source are the cells from the lower intact hair follicle. Th ese Sox9+ cells will fi ll in the empty place in the absence of Nfatc1+ cells. Th is suggests that under the normal conditions there should be an inhibitory signal from the existing SCs to prevent unwanted SC diff erentiation of the surrounding cells.

44 Results and discussion

According to lineage tracing, Nfatc1+ cells are a narrow population in the developing follicle, which contribute to sebaceous gland and hair follicle SCs (Xu et al., 2015), while embryonic Sox9+ cells have a wider diff erentiation potential and contribute to ORS and diff erentiated structures of the hair follicle aft er morphogenesis (Nowak et al., 2008; Xu et al., 2015). Deciphering the diff erentiation potential of Lhx2+ cells would be interesting, but lineage tracing experiments have not been performed yet. In Foxi3 KO hair follicles, Sox9 was not aff ected, and expression of Lhx2 was restored at advanced stages. However, stable downregulation of Nfatc1 in Foxi3 KO indicates that Foxi3 is involved specifi cally in hair follicle SC specifi cation.

4.3.3 Foxi3 is required for hair follicle stem cell maintenance and acƟ vaƟ on To characterise the maintenance of hair follicle SCs upon Foxi3 deletion, we analysed SC markers in Foxi3 cKO at two diff erent stages: the 1st telogen when bulge and hair germ morphologically appeared for the fi rst time, and 1 year old aft er approximately 7-8 cycles of follicle regeneration (Waghmare et al., 2008). By the 1st telogen, many Foxi3 cKO hair follicles displayed depletion of the SC compartments, bulge and hair germ, showed by decreased amount of Sox9 and Lhx2 positive hair follicles and less Lhx2+ cells in the follicles. Th e readout of activated Wnt signaling, Lef-1 (II), and Wnt co-receptor Lgr6 (Fig. 10) were both absent from aff ected Foxi3 cKO hair follicles, suggesting that activation of the Wnt pathway required for proliferation at anagen onset is abolished in Foxi3 cKO. At the age of one year, many more Foxi3 cKO hair follicles exhausted their SC reservoirs and had less Lhx2+ cells compared to the controls and to the Foxi3 cKO 1st telogen follicles.

Control Foxi3 cKO First telogen

Lgr6

Figure 10. Downregulation of Wnt signaling in telogen hair follicle of Foxi3 cKO. Lack of Lgr6 expression in malformed hair follicles of Foxi3 cKO. Lgr6 expression was detected by radioactive ISH at fi rst telogen, P20. Scale bar=50 μm.

Foxi3 expression in the hair germ led us to check whether Foxi3 is directly involved in the activation of follicle regeneration. We challenged Foxi3 cKO hair follicles with hair plucking to activate a new round of hair follicle self-renewal. Foxi3 cKO individuals grew hair back slower due to decreased proliferation. A schematic summary of the role of Foxi3 for specifi cation and maintenance of hair follicle SCs was proposed in the publication (I).

45 Results and discussion

It was shown that Shh produced by TA cells drives proliferation of both bulge and TA cells (Hsu et al., 2014). We wondered whether Foxi3 could regulate generation of Shh-producing TA cells at the beginning of anagen and by that mechanism, indirectly modulate bulge SC proliferation. We analysed Shh expression by radioactive ISH in Foxi3 cKO and found that Foxi3 cKO downregulate Shh expression both in natural fi rst anagen and at depilation-activated regeneration. Th is result was opposite to what was seen in embryonic Foxi3-defi cient hair follicles, where Shh expression was not aff ected. However, it was consistent with the observation of Shh downregulation in developing Foxi3 cKO molar (Jussila et al., 2015). In developing hair follicle, Shh activates Sox9 expression and expansion of SC progenitor cells. Sox9 is downregulated in embryonic hair follicles of mice with germline targeted Shh and Gli2 deletion and in Smo cKO (Vidal et al., 2005; Ouspenskaia et al., 2016), that should lead to failure in SC specifi cation. Hair follicles of mice with conditional deletion of Shh and its downstream components generate shortened follicles during anagen, exhibiting an embryonic downgrowth phenotype. Nevertheless, Shh pathway cKO hair follicles are able to self-renew (Hsu et al., 2014), indicating that Sox9+ bulge and hair germ cells are not severely aff ected, though there are no direct data on Sox9 expression in these Shh cKO mice. Apparently, in the embryonic context, Shh is a more potent signal for the hair follicles than in postnatal skin. Th is also may account for the presence of more pathways that regulate Shh expression in the embryo, and the deletion of a single gene, Foxi3, does not have impact on Shh signaling. Although speculative, it is possible that in postnatal skin, there might be fewer stabilising factors for Shh signaling and less genes that are sensitive to Shh, as could be seen from SC markers that remained present in mice with conditionally ablated Shh signaling. Th is lowers the threshold for sensitivity of Shh pathway to upstream regulation, and Foxi3 ablation is enough to compromise Shh signaling. It was shown previously (Hsu et al., 2011) that the hair follicle SC pool is maintained partly by the progenitor cells that retain their proliferative potential during the hair cycle. Our data suggest that Foxi3 regulates the maintenance of the SC pool and SC activation for regeneration. Since Foxi3 is expressed in the hair germ, Foxi3 might defi ne the proliferation rate and regulate the amount of follicular tissue generated. Th rough this the hair follicle may adjust the replenishment of the SC pool at the end of the cycle, as it was shown that Shh cKO, Gli2 cKO and Smo cKO mice with shortened ORS also have smaller hair germs and impaired anagen activation (Hsu et al., 2014).

4.4 Tsr2 is a novel regulator of hair follicle development and growth A congenital hairless condition could be driven by mutation in a vast number of genes (Lemke et al., 2013), and many hair disorder phenotypes are still awaiting detection of the causative mutation. During the progress of this PhD thesis work, one more gene behind hairless phenotype was identifi ed. Female cows of Pezzata Rossa breed carry Z-shaped hairless streaks in diff erent parts of the body (Wollenberg and Eames, 2011). No aff ected bulls were found, suggesting that in males, the mutation is lethal. Hairless sites of the skin lack hair follicles and sebaceous glands, while sweat glands were present. Genotype comparison of four aff ected females against eight unaff ected related cows revealed 777,762 single nucleotide polymorphisms (SNP). Haplotype analysis

46 Results and discussion showed that all four aff ected cows shared one disease-linked haplotype spanning the entire X-chromosome, confi rming X-linked inheritance of the trait. Whole genome sequencing of one aff ected cow and linkage analysis identifi ed mutations in two candidate genes: the excision repair cross-complementation group 6-like (Ercc6l), a DNA helicase, and 20S rRNA accumulation, homolog (S. cerevisiae) (Tsr2). In order to evaluate the potential involvement of these genes in the hairless phenotype, we studied their expression in murine hair follicles. Further analysis of Ercc6l showed that a missense mutation in Ercc6l localized in a non- conserved region and was predicted to lead to a non-damaging amino acid change. We did not detect Ercc6l expression in developing and adult hair follicles. Collectively, these data argue that Ercc6l is not the causative gene. Th e other variant was a point mutation aff ecting the 5’-splice junction of exon 5 of the Tsr2 gene, resulting in the formation of a C-terminally truncated version of the protein. Mutant transcripts of Tsr2 accumulated in the skin of aff ected cows. Whole mount and radioactive ISH detected Tsr2 expression in the epithelium of developing hair follicle and in the growing portion of the regenerating hair follicle. Th e level of expression was low but distinguishable from the sense probe control. Tsr2 is a poorly characterised gene. In humans, mutation in TSR2 has been associated with Diamond-Blackfan anemia (Gripp et al., 2014). Out of 16 causative genes, TSR2 and GATA1 are located on the X-chromosome, and TSR2-associated anemia represent a rare group of X-linked inherited anemias (Clinton and Gazda, 2009). TSR2 mutation in the WGG domain of unknown function was identifi ed in two aff ected cousins. Th eir mothers were sisters and were healthy (Gripp et al., 2014). Tsr2 mutation in cows is not in the WGG domain and apparently is more severe since the aff ected bulls most likely are not viable. Even though in some cases of Diamond- Blackfan anemia, a partial loss of eyebrows and lashes was described (Gripp et al., 2014), Tsr2 has not yet been connected to hair follicle development and regeneration. Overall, the function of Tsr2 is poorly understood. Some evidence suggests its involvement in 20S rRNA processing and further maturation of ribosomal subunits. One can speculate that mutation in the regulator of ribosomal biogenesis might deleteriously aff ect highly proliferative organs such as hair follicles. Defects in cellular proliferation might lead to impaired growth, which ultimately results in complete lack of follicles in the aff ected skin. Tsr2, as a ribosomal regulator, should be expressed rather uniformly in all tissues, while its deleterious eff ect targeted the hair follicles only. Interestingly, certain hair defects were shown to be caused by mutations in ubiquitously expressed genes. Two of small nuclear ribonucleo proteins (snRNP): RBM28 and SNRPE, have been associated with cases of hypotrichosis, and mutation in DCAF17 which encodes a nucleolar protein with unknown function, causes a complex syndrome which also features alopecia (Duverger and Morasso, 2014). Restriction of the deleterious eff ect of Tsr2 mutation to skin might represent lack of a compensatory mechanism in the skin or, alternatively, selective survival of the cells carrying the mutation exclusively in the skin. In essence, lines of Blaschko of Pezzata Rossa cows caused by mutation in Tsr2 is a novel monogenic animal hair disorder.

47 Conclusion and future perspectives 5 CONCLUSION AND FUTURE PERSPECTIVES

In this PhD thesis the role of Foxi3 during diff erent aspects of hair follicle development and postnatal regeneration was elucidated. However, certain questions on SC specifi cation and Foxi3 action as a transcription factor remained outside of this study. Foxi3 is expressed early in hair development in the leading edge of the growing hair follicle and is colocalized with SC marker Lhx2. Basal cells of the growing placode express elevated levels of Wnt signaling (Ouspenskaia et al., 2016). Suprabasal cells attenuate Wnt, gain Sox9 expression and become SC precursors (Ouspenskaia et al., 2016; Xu et al., 2015). It would be interesting to look at the Wnthi population in Foxi3-defi cient background and to examine the outcome of Foxi3 loss on the balance of distinct cell populations in bud stage follicles. Foxi3 expression changes dynamically; from the leading edge of the hair germ to the central cells of the growing follicle and fi nally it is expressed in the secondary hair germ at telogen. Apparently, diff erent Foxi3+ cells contribute to the distinct cell lineages of postnatal hair follicle. To comprehensively answer the question on the identity of Foxi3+ cells, lineage tracing of Foxi3 descendants starting from diff erent time points would be desired, and this would require creation of a Foxi3-CreER mouse. Foxi3 is one of the few genes specifi cally expressed in the secondary hair germ SCs and thus could be used as a cell marker for this population. Cell sorting techniques require such specifi c, highly expressed markers. Hair follicle SCs could be FACS isolated in suffi cient quantities for diff erent manipulations, such as gene expression and ChIP analysis, in vitro expansion, and others. However, separation between bulge and hair germ SC populations is challenging. Foxi3 might be a useful tool to determine the purity of the isolated hair germ cells. As a transcription factor, Foxi3 activates the expression of several SC related genes. ChIP- sequencing would be helpful to fi nd out whether these are direct Foxi3 target genes whose enhancers/promoters are occupied by Foxi3. Several commercial Foxi3 antibodies became available during the course of this study, but whether any of them work in ChIP remains to be tested. Another interesting direction is the possible role of Foxi3 as a pioneer transcription factor. Zebrafi sh Foxi1 was shown to act as a pioneer factor (Yan et al., 2006). Foxi3 expression pattern and Foxi3-defi cient otic phenotypes in chicken and mouse suggest that Foxi3 functions as early pioneer factor for the otic lineage during ear induction (Edlund et al., 2014; Khatri et al., 2014). One of the genes downregulated upon Foxi3 loss is Runx1, which is responsible for hair follicle SC specifi cation at embryogenesis and SC activation at regeneration (Osorio et al., 2011). However, Runx1 alone is not suffi cient for initiation of anagen, demanding for additional promoting stimuli (Hoi et al., 2010). Th e postnatal hair phenotype of Foxi3-defi cient mice suggests that Foxi3 is essential for SC activation at the beginning of the new growth. Given the parallels in outcomes of the gene loss for the hair follicle SCs, Foxi3 might act in cooperation with Runx1, activating gene expression. Another Fox transcription factor, Foxp3, was shown to directly interact with Runx1 in regulatory T cells (T(R) cells) and mediate transcription of T(R) cell-associated genes conferring suppressive activity for T(R) cells in regulating of excessive immune responses (Ono et al., 2007).

48 Conclusion and future perspectives

Recently, superenhancers were described as regulators of cellular diff erentiation in the hair follicle (Adam et al., 2015). In the hair follicle SCs, super-enhancers are occupied by Sox9, Nfatc1, Lhx2, Tcf3/4. Foxi3, in cooperation with Lhx2 and Nfatc1, might act through binding to superenhancers of the hair germ SCs to regulate the expression of a cohort of genes and promote cell fate diff erentiation. Mutation of Foxi3 drives a condition named canine ED (Drögemuller et al., 2008). Th e most common cause of HED in many species is defi ciency in one of the components of the Eda pathway (Mikkola, 2009). Using mouse models with altered Eda expression and an in vitro approach of treating samples with recombinant Eda, we have demonstrated that Eda regulates Foxi3. Th us, Foxi3 might be a candidate gene for those ectodermal organ diseases where a mutation in the Eda pathway has not been identifi ed. In addition to ectodermal appendage development, Foxi3 controls ear development (Edlund et al., 2014). A documented case of congenital aural atresia, microtia, and ipsilateral internal carotid artery agenesis in a female baby was associated with FOXI3 deletion (Tassano et al., 2015). Th erefore, Foxi3 might represent of potential interest as a causative gene of congenital hair disorders. Study of a hair disorder condition of the cow revealed a new gene with a function in the hair follicle, Tsr2. Th e expression pattern of Tsr2 suggests that it is activated during embryonic and postnatal follicle growth. However, more detailed studies are needed to clarify the role for Tsr2 in hair follicle biology, the fi rst step being the generation of conditional mutant mice with targeted Tsr2 ablation in skin. Th e hair follicle represents a great model for developmental biology research, starting from morphogenetic events based on crosstalk between the signalling pathways and all the way to SC function in the adulthood. Here, the role for Foxi3 in the regulation of hair follicle SCs was shown, answering some, but raising new questions on the biology of the hair follicle.

49 Acknowledgements 6 ACNOWLEDGEMENTS

Th is thesis work was carried out in the Institute of Biotechnology in the University of Helsinki with the fi nancial support from the Viikki Doctoral Programme in Molecular Biosciences, the Integrative Life Science (ILS) doctoral program and the Ella and Georg Ehrnrooth foundation. I wish to acknowledge the former directors of the Institute, Professor Mart Saarma and Professor Tomi Mäkelä, and the present director Howard Jacobs for providing excellent research facilities. I wish to express my sincere gratitude to my supervisor, Docent Marja Mikkola. Th ank you for your everyday williness to share your deep knowledge and experience, for impetuous e-mail replies and for giving a feeling of secure and confi dence, which are so important for carrying out the research work. Your personal involvement and enthusiasm towards the science were the source of inspiration for the research work that we are doing. I also wish to express my deepest gratitude and respect to Professor Irma Th esleff . It was an honor to work close to you and to be taught by your personality how the highest standard in science goes along with humane and promoting atmosphere in the laboratory. I thank the members of my follow-up group, Docent Ulla Pirvola and Professor Antti IIvanainen for the fruitful work at the annual Th esis Committee meeting and for your interest to my work and life though all these years. I also wish to thank Professor Antti Iivanainen and Professor Seppo Vainio for the careful review and discussion of my thesis. I acknowledge Professor Vladimir Botchkarev for accepting invitation to act as my opponent at the public defense of the thesis. I am grateful to our collaborators and co-authors, especially Andrew Groves, Marjo Hyttönen, Tosso Leeb and Leonardo Murgiano for their work and advice. I thank Timo Paivarinta for the layout of the thesis and the swift printing of my posters, and Anita Tienhaara for the cover layout. Every spring and autumn, my heart will be calling me to Tvarminne and Hyytiälä meetings. Th ank you to all Helsinki Developmental Biologists and all the members of the Finnish Society for Developmental Biology for the great time! I am especially grateful to Professor Scott Gilbert, for amazing lectures at Halloween and at other events, for the deepest expertise in Developmental Biology that he is ready to share with the students, and for endless curious stories from scientifi c and non-scientifi c fi elds. I have also gained a lot from the courses and events organized by VGSB and ILS, for what I wish to acknowledge our coordinator Erkki Raulo. I was also given a chance to be on the other side of the teaching process and assist at the course “Methods in Functional Genetics and Development”. I enjoyed this work a lot and wish to thank the organizer of the course, Professor Juha Partanen, and my supervisor Marja Mikkola for giving me an opportunity to teach there. Th ese PhD years gave me a great luck of working with amazing talented people who were not only my co-workers and collaborators but also became my second family here away of home. My two dear Marias, Meeri Jussila and Maria Voutilanen, you are the people whom I will always remember whenever I think about my lab years. I cannot imagine my work without you. I wish to thank Katja Närhi, Sylvie Lefebvre and Otso Häärä for being helpful and cheering up at the beginning of my path. I am very thankful to all the present lab members. Th ank you for the nice atmosphere at the bench and at the desk to my old and new offi ce-mates, Emma, Fred, Ana-Marija, Ewelina and Otto. I wish to thank my senior colleagues who were always ready to help with the discussion and advice, Elodie Renvoise, Laura Ahtianen, Frederic Michon and especially charismatic and

50 Acknowledgements supportive Ana Balic. Riitta and Qiang, without your technical help not only my thesis would not be fi nished, but hardly any of my computer fi ght would be won! Teresa, you are the best listener and supporter in all my child-related worries. My dear girls from Fred’s lab, Maria and Solja, thank you for all the help and support that you are giving me both in and outside the lab! I wish to acknowledge Leah Biggs for the proofreading of this thesis book, as well as for your supportive friendship and our scientifi c and family-related chats. Th e biggest thank you goes to our lab moms, our technicians Raija Savolainen, Merja Makinen, and Riikka Santalahti, for all your work, help, wisdom and patience. Th e lab would not ran without you any single day! Dear Raija, I was lucky to have the neighboring bench with you, and your support and wise words oft en fueled the forces for another one experiment. I wish to thank my great friends that were with me through these years. My dear Helsinki team, Sveta, Kolja, Ilya, Andrei, Ruslan, Dima, Lena, Tonja, Jenya, your help and support are invaluable for me. Th ank you to amazing Maarja Laos for her inspirative attitude. My St-Petersburg duo, Julia and Marina, it is wonderful that we are keeping together since University times. Th e single non-biologist in my life, Natasha, thank you so very much for all those 25 or so years of friendship fi lled with many unforgettable moments, and for being a person who listens and says exactly those words that help to keep going. My new hobbies, dancing and Unisport, fostered my energy for the lab work and scientifi c thinking. My precious teachers and friends, Johanna, Omar, Masha, Mira Ravald, Guillermo Sarduy and Emilia Lindström, thank you for giving me strength and inspiration and for opening for me the world of non-verbal expression. I am grateful to my dear family, my mom and dad and my brother’s family, for the interest towards my work and for the support during the years. Dear Lena, Leva, and my beloved nephews and niece, Maxim, Kirill and Dasha, I hope we will have more nice family moments together. Nikolai Aleksandrovich, thank you that some time ago you paved my way to studying biology. At the end, I wish to thank the most important people in my life, my mom and my son. Dear mom, having your care at the back means everything for me, thank you for your patience, support and acceptance of what am I, and for babysitting in all those moments when my mind was completely captured by thesis work. All this would not happen without your help. Sasha, thank you for being a miracle in my life and for letting me to experience totally new things. You are my best, the most beloved and never ending Developmental Biology project.

Helsinki, October 2016

Vera Shirokova

51 References 7 REFERENCES

Adam, R.C., and Fuchs, E. (2016). Th e Yin and Yang Bhakar, A.L., Tannis, L.L., Zeindler, C., Russo, M.P., of Chromatin Dynamics In Stem Cell Fate Selection. Jobin, C., Park, D.S., MacPherson, S., and Barker, P.A. Trends Genet. 32, 89-100. (2002). Constitutive nuclear factor-kappa B activity is required for central neuron survival. J. Neurosci. 22, Adam, R.C., Yang, H., Rockowitz, S., Larsen, S.B., 8466-8475. Nikolova, M., Oristian, D.S., Polak, L., Kadaja, M., Asare, A., Zheng, D., and Fuchs, E. (2015). Pioneer Bianchi, N., Depianto, D., McGowan, K., Gu, C., and factors govern super-enhancer dynamics in stem cell Coulombe, P.A. (2005). Exploiting the gene plasticity and lineage choice. Nature 521, 366-370. promoter to visualize live cells in epithelial appendag- es of mice. Mol. Cell. Biol. 25, 7249-7259. Ahmad, W., Faiyaz ul Haque, M., Brancolini, V., Tsou, H.C., ul Haque, S., Lam, H., Aita, V.M., Owen, J., de- Biggs, L.C., and Mikkola, M.L. (2014). Early inductive Blaquiere, M., Frank, J., et al. (1998). Alopecia univer- events in ectodermal appendage morphogenesis. Se- salis associated with a mutation in the human hairless min. Cell Dev. Biol. 25-26, 11-21. gene. Science 279, 720-724. Blanpain, C., Lowry, W.E., Geoghegan, A., Polak, L., Ahn, Y. (2015). Signaling in tooth, hair, and mamma- and Fuchs, E. (2004). Self-renewal, multipotency, and ry placodes. Curr. Top. Dev. Biol. 111, 421-459. the existence of two cell populations within an epithe- lial stem cell niche. Cell 118, 635-648. Ahn, Y., Sims, C., Logue, J.M., Weatherbee, S.D., and Krumlauf, R. (2013). Lrp4 and Wise interplay con- Bluschke, G., Nusken, K.D., and Schneider, H. (2010). trols the formation and patterning of mammary and Prevalence and prevention of severe complications of other skin appendage placodes by modulating Wnt hypohidrotic ectodermal dysplasia in infancy. Early signaling. Development 140, 583-593. Hum. Dev. 86, 397-399. Ahtiainen, L., Lefebvre, S., Lindfors, P.H., Renvoise, Bohring, A., Stamm, T., Spaich, C., Haase, C., Spree, E., Shirokova, V., Vartiainen, M.K., Th esleff , I., and K., Hehr, U., Hoff mann, M., Ledig, S., Sel, S., Wieack- Mikkola, M.L. (2014). Directional cell migration, but er, P., and Ropke, A. (2009). WNT10A mutations are not proliferation, drives hair placode morphogenesis. a frequent cause of a broad spectrum of ectodermal Dev. Cell. 28, 588-602. dysplasias with sex-biased manifestation pattern in heterozygotes. Am. J. Hum. Genet. 85, 97-105. Andl, T., Ahn, K., Kairo, A., Chu, E.Y., Wine-Lee, L., Reddy, S.T., Croft , N.J., Cebra-Th omas, J.A., Metzger, Bollenbach, T., Pantazis, P., Kicheva, A., Bokel, C., D., Chambon, P., et al. (2004). Epithelial Bmpr1a reg- Gonzalez-Gaitan, M., and Julicher, F. (2008). Preci- ulates diff erentiation and proliferation in postnatal sion of the Dpp gradient. Development 135, 1137- hair follicles and is essential for tooth development. 1146. Development 131, 2257-2268. Botchkarev, V.A. (2003). Bone morphogenetic pro- Andl, T., Reddy, S.T., Gaddapara, T., and Millar, S.E. teins and their antagonists in skin and hair follicle (2002). WNT signals are required for the initiation of biology. J. Invest. Dermatol. 120, 36-47. hair follicle development. Dev. Cell. 2, 643-653. Botchkarev, V.A., Botchkareva, N.V., Roth, W., Na- Angier, N. (2003). Do Straw Men Have DNA? Th e kamura, M., Chen, L.H., Herzog, W., Lindner, G., American Scholar Spring, McMahon, J.A., Peters, C., Lauster, R., McMahon, A.P., and Paus, R. (1999). Noggin is a mesenchymally Arora, M., Zhang, J., Heine, G.F., Ozer, G., Liu, H.W., derived stimulator of hair-follicle induction. Nat. Cell Huang, K., and Parvin, J.D. (2012). Promoters ac- Biol. 1, 158-164. tive in interphase are bookmarked during mitosis by ubiquitination. Nucleic Acids Res. 40, 10187-10202. Botchkarev, V.A., and Fessing, M.Y. (2005). Edar sig- naling in the control of hair follicle development. J. Aubin-Houzelstein, G. (2012). Notch signaling and Investig. Dermatol. Symp. Proc. 10, 247-251. the developing hair follicle. Adv. Exp. Med. Biol. 727, 142-160. Brownell, I., Guevara, E., Bai, C.B., Loomis, C.A., and Joyner, A.L. (2011). Nerve-derived sonic hedgehog Bazzi, H., Fantauzzo, K.A., Richardson, G.D., Jahoda, defi nes a niche for hair follicle stem cells capable of C.A., and Christiano, A.M. (2007). Th e Wnt inhibitor, becoming epidermal stem cells. Cell. Stem Cell. 8, Dickkopf 4, is induced by canonical Wnt signaling 552-565. during ectodermal appendage morphogenesis. Dev. Biol. 305, 498-507.

52 References

Brownell, I., Guevara, E., Bai, C.B., Loomis, C.A., and Clarke, A., Phillips, D.I., Brown, R., and Harper, P.S. Joyner, A.L. (2011). Nerve-derived sonic hedgehog (1987). Clinical aspects of X-linked hypohidrotic ec- defi nes a niche for hair follicle stem cells capable of todermal dysplasia. Arch. Dis. Child. 62, 989-996. becoming epidermal stem cells. Cell. Stem Cell. 8, Clavel, C., Grisanti, L., Zemla, R., Rezza, A., Barros, 552-565. R., Sennett, R., Mazloom, A.R., Chung, C.Y., Cai, X., Casal, M.L., Lewis, J.R., Mauldin, E.A., Tardivel, A., Cai, C.L., et al. (2012). Sox2 in the dermal papilla Ingold, K., Favre, M., Paradies, F., Demotz, S., Gaide, niche controls hair growth by fi ne-tuning BMP sig- O., and Schneider, P. (2007). Signifi cant correction of naling in diff erentiating hair shaft progenitors. Dev. disease aft er postnatal administration of recombinant Cell. 23, 981-994. ectodysplasin A in canine X-linked ectodermal dys- Clinton, C., and Gazda, H.T. (1993). Diamond-Black- plasia. Am. J. Hum. Genet. 81, 1050-1056. fan Anemia. In GeneReviews(R), Pagon, R. A., Adam, Casal, M.L., Scheidt, J.L., Rhodes, J.L., Henthorn, M. P., Ardinger, H. H., Wallace, S. E., Amemiya, A., P.S., and Werner, P. (2005). Mutation identifi cation Bean, L. J. H., Bird, T. D., Fong, C. T., Meff ord, H. C., in a canine model of X-linked ectodermal dysplasia. Smith, R. J. H., et al. eds., (Seattle (WA): University of Mamm. Genome 16, 524-531. Washington, Seattle) Chen, C.C., Wang, L., Plikus, M.V., Jiang, T.X., Mur- Cotsarelis, G., Sun, T.T., and Lavker, R.M. (1990). La- ray, P.J., Ramos, R., Guerrero-Juarez, C.F., Hughes, bel-retaining cells reside in the bulge area of piloseba- M.W., Lee, O.K., Shi, S., et al. (2015). Organ-level ceous unit: implications for follicular stem cells, hair quorum sensing directs regeneration in hair stem cell cycle, and skin carcinogenesis. Cell 61, 1329-1337. populations. Cell 161, 277-290. Cowin, P., and Wysolmerski, J. (2010). Molecular Chen, D., Jarrell, A., Guo, C., Lang, R., and Atit, R. mechanisms guiding embryonic mammary gland (2012). Dermal beta-catenin activity in response to development. Cold Spring Harb Perspect. Biol. 2, epidermal Wnt ligands is required for fi broblast pro- a003251. liferation and hair follicle initiation. Development Crabtree, G.R., and Graef, I.A. (2008). Bursting into 139, 1522-1533. the nucleus. Sci. Signal. 1, pe54. Chen, T., Heller, E., Beronja, S., Oshimori, N., Stokes, Crabtree, G.R., and Olson, E.N. (2002). NFAT signal- N., and Fuchs, E. (2012). An RNA interference screen ing: choreographing the social lives of cells. Cell 109 uncovers a new molecule in stem cell self-renewal and Suppl, S67-79. long-term regeneration. Nature 485, 104-108. Croyle, M.J., Lehman, J.M., O’Connor, A.K., Wong, Chi, W., Wu, E., and Morgan, B.A. (2015). Earli- S.Y., Malarkey, E.B., Iribarne, D., Dowdle, W.E., er-born secondary hair follicles exhibit phenotypic Schoeb, T.R., Verney, Z.M., Athar, M., et al. (2011). plasticity. Exp. Dermatol. 24, 265-268. Role of epidermal primary cilia in the homeostasis of Chi, W., Wu, E., and Morgan, B.A. (2013). Dermal skin and hair follicles. Development 138, 1675-1685. papilla cell number specifi es hair size, shape and cy- DasGupta, R., and Fuchs, E. (1999). Multiple roles for cling and its reduction causes follicular decline. De- activated LEF/TCF transcription complexes during velopment 140, 1676-1683. hair follicle development and diff erentiation. Devel- Chiang, C., Swan, R.Z., Grachtchouk, M., Bolinger, opment 126, 4557-4568. M., Litingtung, Y., Robertson, E.K., Cooper, M.K., Deschene, E.R., Myung, P., Rompolas, P., Zito, G., Sun, Gaffi eld, W., Westphal, H., Beachy, P.A., and Dlugosz, T.Y., Taketo, M.M., Saotome, I., and Greco, V. (2014). A.A. (1999). Essential role for Sonic hedgehog during beta-Catenin activation regulates tissue growth non- hair follicle morphogenesis. Dev. Biol. 205, 1-9. cell autonomously in the hair stem cell niche. Science Choi, Y.S., Zhang, Y., Xu, M., Yang, Y., Ito, M., Peng, 343, 1353-1356. T., Cui, Z., Nagy, A., Hadjantonakis, A.K., Lang, R.A., Driskell, R.R., Giangreco, A., Jensen, K.B., Mulder, et al. (2013). Distinct functions for Wnt/beta-catenin K.W., and Watt, F.M. (2009). Sox2-positive dermal in hair follicle stem cell proliferation and survival and papilla cells specify hair follicle type in mammalian interfollicular epidermal homeostasis. Cell. Stem Cell. epidermis. Development 136, 2815-2823. 13, 720-733. Drogemuller, C., Distl, O., and Leeb, T. (2001). Par- Cirillo, L.A., and Barton, M.C. (2008). Many fork- tial deletion of the bovine ED1 gene causes anhidrotic heads in the road to regulation. Symposium on fork- ectodermal dysplasia in cattle. Genome Res. 11, 1699- head transcription factor networks in development, 1705. signalling and disease. EMBO Rep. 9, 721-724.

53 References

Drogemuller, C., Karlsson, E.K., Hytonen, M.K., Per- Folgueras, A.R., Guo, X., Pasolli, H.A., Stokes, N., Po- loski, M., Dolf, G., Sainio, K., Lohi, H., Lindblad-Toh, lak, L., Zheng, D., and Fuchs, E. (2013). Architectural K., and Leeb, T. (2008). A mutation in hairless dogs niche organization by LHX2 is linked to hair follicle implicates FOXI3 in ectodermal development. Sci- stem cell function. Cell. Stem Cell. 13, 314-327. ence 321, 1462. Frank, J., Pignata, C., Panteleyev, A.A., Prowse, D.M., Dry F W. (1926). Th e coat of the mouse (Mus muscu- Baden, H., Weiner, L., Gaetaniello, L., Ahmad, W., lus). J Genet 16, :287-340. Pozzi, N., Cserhalmi-Friedman, P.B., et al. (1999). Exposing the human nude phenotype. Nature 398, Duverger, O., and Morasso, M.I. (2014). To grow or 473-474. not to grow: hair morphogenesis and human genetic hair disorders. Semin. Cell Dev. Biol. 25-26, 22-33. French, C.A., and Fisher, S.E. (2014). What can mice tell us about Foxp2 function? Curr. Opin. Neurobiol. Duverger, O., and Morasso, M.I. (2009). Epider- 28, 72-79. mal patterning and induction of diff erent hair types during mouse embryonic development. Birth Defects Fuchs, E. (2016). Epithelial Skin Biology: Th ree De- Res. C. Embryo. Today 87, 263-272. cades of Developmental Biology, a Hundred Ques- tions Answered and a Th ousand New Ones to Ad- Edlund, R.K., Ohyama, T., Kantarci, H., Riley, B.B., dress. Curr. Top. Dev. Biol. 116, 357-374. and Groves, A.K. (2014). Foxi transcription factors promote pharyngeal arch development by regulating Fujimoto, A., Farooq, M., Fujikawa, H., Inoue, A., formation of FGF signaling centers. Dev. Biol. 390, Ohyama, M., Ehama, R., Nakanishi, J., Hagihara, 1-13. M., Iwabuchi, T., Aoki, J., Ito, M., and Shimomura, Y. (2012). A missense mutation within the helix ini- Eldridge, F., and Atkeson, F. (1953). Streaked hair- tiation motif of the keratin K71 gene underlies auto- lessnss in Holstein Friesian cattle. Journal of Heredity somal dominant woolly hair/hypotrichosis. J. Invest. 44, 265. Dermatol. 132, 2342-2349. Enshell-Seijff ers, D., Lindon, C., Kashiwagi, M., and Fukuta, K., Koizumi, N., Imamura, K., Goto, N., and Morgan, B.A. (2010). Beta-Catenin Activity in the Hamada, H. (1991). Microscopic observations of skin Dermal Papilla Regulates Morphogenesis and Regen- and lymphoid organs in the hairless dog derived from eration of Hair. Dev. Cell. 18, 633-642. the Mexican hairless. Jikken Dobutsu 40, 69-76. Falconer, D. C. (1952). A totally sex-linked gene in the Gaide, O., and Schneider, P. (2003). Permanent cor- house mouse. Nature 169, 664-665. rection of an inherited ectodermal dysplasia with re- Fang, J., Dagenais, S.L., Erickson, R.P., Arlt, M.F., combinant EDA. Nat. Med. 9, 614-618. Glynn, M.W., Gorski, J.L., Seaver, L.H., and Glover, Garza, L.A., Liu, Y., Yang, Z., Alagesan, B., Lawson, T.W. (2000). Mutations in FOXC2 (MFH-1), a fork- J.A., Norberg, S.M., Loy, D.E., Zhao, T., Blatt, H.B., head family transcription factor, are responsible for Stanton, D.C., et al. (2012). Prostaglandin D2 inhibits the hereditary lymphedema-distichiasis syndrome. hair growth and is elevated in bald scalp of men with Am. J. Hum. Genet. 67, 1382-1388. androgenetic alopecia. Sci. Transl. Med. 4, 126ra34. Fantauzzo, K.A., Kurban, M., Levy, B., and Christiano, Gat, U., DasGupta, R., Degenstein, L., and Fuchs, E. A.M. (2012). Trps1 and its target gene Sox9 regulate (1998). De Novo hair follicle morphogenesis and hair epithelial proliferation in the developing hair follicle tumors in mice expressing a truncated beta-catenin in and are associated with hypertrichosis. PLoS Genet. skin. Cell 95, 605-614. 8, e1003002. Gelperin, D., Horton, L., Beckman, J., Hensold, J., and Fessing, M.Y., Sharova, T.Y., Sharov, A.A., Atoyan, R., Lemmon, S.K. (2001). Bms1p, a novel GTP-binding and Botchkarev, V.A. (2006). Involvement of the Edar protein, and the related Tsr1p are required for dis- signaling in the control of hair follicle involution (cat- tinct steps of 40S ribosome biogenesis in yeast. Rna agen). Am. J. Pathol. 169, 2075-2084. 7, 1268-1283. Fliniaux, I., Mikkola, M.L., Lefebvre, S., and Th esleff Genander, M., Cook, P.J., Ramskold, D., Keyes, B.E., I. (2008). Identifi cation of dkk4 as a target of Eda-A1/ Mertz, A.F., Sandberg, R., and Fuchs, E. (2014). BMP Edar pathway reveals an unexpected role of ectodys- signaling and its pSMAD1/5 target genes diff erential- plasin as inhibitor of Wnt signalling in ectodermal ly regulate hair follicle stem cell lineages. Cell. Stem placodes.. Dev Biol. 320, 60-71. Cell. 15, 619-633.

54 References

Geyfman, M., Plikus, M.V., Treff eisen, E., Andersen, Headon, D.J., Emmal, S.A., Ferguson, B.M., Tucker, B., and Paus, R. (2015). Resting no more: re-defi ning A.S., Justice, M.J., Sharpe, P.T., Zonana, J., and Over- telogen, the maintenance stage of the hair growth cy- beek, P.A. (2001). Gene defect in ectodermal dysplasia cle. Biol. Rev. Camb. Philos. Soc. 90, 1179-1196. implicates a death domain adapter in development. Nature 414, 913-916. Gilbert, S., Fidgerald. (2014). Developmental Biology (Massachusetts USA: Sinauer Associates Inc. Sunder- Headon, D.J., and Overbeek, P.A. (1999). Involvement land). of a novel Tnf receptor homologue in hair follicle in- duction. Nat. Genet. 22, 370-374. Goldstein, J., and Horsley, V. (2012). Home sweet home: skin stem cell niches. Cell Mol. Life Sci. 69, Higgins, C.A., Westgate, G.E., and Jahoda, C.A. 2573-2582. (2009). From telogen to exogen: mechanisms under- lying formation and subsequent loss of the hair club Goto, N., Imamura, K., Miura, Y., Ogawa, T., and fi ber. J. Invest. Dermatol. 129, 2100-2108. Hamada, H. (1987). Th e Mexican hairless dog, its morphology and inheritance. Jikken Dobutsu 36, 87- Hoi, C.S., Lee, S.E., Lu, S.Y., McDermitt, D.J., Osorio, 90. K.M., Piskun, C.M., Peters, R.M., Paus, R., and Tum- bar, T. (2010). Runx1 directly promotes proliferation Greco, V., Chen, T., Rendl, M., Schober, M., Pasolli, of hair follicle stem cells and epithelial tumor forma- H.A., Stokes, N., Dela Cruz-Racelis, J., and Fuchs, E. tion in mouse skin. Mol. Cell. Biol. 30, 2518-2536. (2009). A two-step mechanism for stem cell activation during hair regeneration. Cell. Stem Cell. 4, 155-169. Horsley, V., Aliprantis, A.O., Polak, L., Glimcher, L.H., and Fuchs, E. (2008). NFATc1 balances quiescence Gripp, K.W., Curry, C., Olney, A.H., Sandoval, C., and proliferation of skin stem cells. Cell 132, 299-310. Fisher, J., Chong, J.X., UW Center for Mendelian Ge- nomics, Pilchman, L., Sahraoui, R., Stabley, D.L., and Houghton, L., Lindon, C.M., Freeman, A., and Mor- Sol-Church, K. (2014). Diamond-Blackfan anemia gan, B.A. (2007). Abortive placode formation in the with mandibulofacial dystostosis is heterogeneous, feather tract of the scaleless chicken embryo. Dev. including the novel DBA genes TSR2 and RPS28. Am. Dyn. 236, 3020-3030. J. Med. Genet. A. 164A, 2240-2249. Hsu, Y.C., Li, L., and Fuchs, E. (2014). Emerging in- Guo, L., Yu, Q.C., and Fuchs, E. (1993). Targeting teractions between skin stem cells and their niches. expression of keratinocyte growth factor to keratino- Nat. Med. 20, 847-856. cytes elicits striking changes in epithelial diff erentia- Hsu, Y.C., Li, L., and Fuchs, E. (2014). Transit-am- tion in transgenic mice. Embo j. 12, 973-986. plifying cells orchestrate stem cell activity and tissue Hans, S., Christison, J., Liu, D., and Westerfi eld, M. regeneration. Cell 157, 935-949. (2007). Fgf-dependent otic induction requires com- Hsu, Y.C., Pasolli, H.A., and Fuchs, E. (2011). Dynam- petence provided by Foxi1 and Dlx3b. BMC Dev. Biol. ics between stem cells, niche, and progeny in the hair 7, 5. follicle. Cell 144, 92-105. Harrington, A.E., Morris-Triggs, S.A., Ruotolo, Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G., B.T., Robinson, C.V., Ohnuma, S., and Hyvönen, M. and Birchmeier, W. (2001). beta-Catenin controls hair (2006). Harrington, A. E. et al. Structural basis for the follicle morphogenesis and stem cell diff erentiation in inhibition of activin signalling by follistatin. Embo j. the skin. Cell 105, 533-545. 25, 1035-1045. Hughes, M.W., Wu, P., Jiang, T.X., Lin, S.J., Dong, Harris, M.P., Rohner, N., Schwarz, H., Perathoner, S., C.Y., Li, A., Hsieh, F.J., Widelitz, R.B., and Chuong, Konstantinidis, P., and Nusslein-Volhard, C. (2008). C.M. (2011). In search of the Golden Fleece: unrav- Zebrafi sh eda and edar mutants reveal conserved and eling principles of morphogenesis by studying the ancestral roles of ectodysplasin signaling in verte- integrative biology of skin appendages. Integr. Biol. brates. PLoS Genet. 4, e1000206. (Camb) 3, 388-407. Harvey, S.A., and Smith, J.C. (2009). Visualisation and Huh, S.H., Narhi, K., Lindfors, P.H., Haara, O., Yang, quantifi cation of morphogen gradient formation in L., Ornitz, D.M., and Mikkola, M.L. (2013). Fgf20 the zebrafi sh. PLoS Biol. 7, e1000101. governs formation of primary and secondary dermal condensations in developing hair follicles. Genes Dev. 27, 450-458.

55 References

Huttner, K. (2014). Future developments in XLHED Kandyba, E., and Kobielak, K. (2014). Wnt7b is an treatment approaches. Am. J. Med. Genet. A. 164A, important intrinsic regulator of hair follicle stem cell 2433-2436. homeostasis and hair follicle cycling. Stem Cells 32, 886-901. Iida, Y., Hibiya, K., Inohaya, K., and Kudo, A. (2014). Eda/Edar signaling guides fi n ray formation with pre- Kandyba, E., Leung, Y., Chen, Y.B., Widelitz, R., ceding osteoblast diff erentiation, as revealed by anal- Chuong, C.M., and Kobielak, K. (2013). Competitive yses of the medaka all-fi n less mutant afl . Dev. Dyn. balance of intrabulge BMP/Wnt signaling reveals a 243, 765-777. robust gene network ruling stem cell homeostasis and cyclic activation. Proc. Natl. Acad. Sci. U. S. A. 110, Itin, P.H. (2013). Ectodermal dysplasia: thoughts and 1351-1356. practical concepts concerning disease classifi cation - the role of functional pathways in the molecular ge- Kaufman, C.K., Zhou, P., Pasolli, H.A., Rendl, M., Bo- netic diagnosis. Dermatology 226, 111-114. lotin, D., Lim, K.C., Dai, X., Alegre, M.L., and Fuchs, E. (2003). GATA-3: an unexpected regulator of cell Ito, M., Yang, Z., Andl, T., Cui, C., Kim, N., Millar, lineage determination in skin. Genes Dev. 17, 2108- S.E., and Cotsarelis, G. (2007). Wnt-dependent de 2122. novo hair follicle regeneration in adult mouse skin aft er wounding. Nature 447, 316-320. Kazantseva, A., Goltsov, A., Zinchenko, R., Grigoren- ko, A.P., Abrukova, A.V., Moliaka, Y.K., Kirillov, A.G., Itoh, N., Nakayama, Y., and Konishi, M. (2016). Roles Guo, Z., Lyle, S., Ginter, E.K., and Rogaev, E.I. (2006). of FGFs As Paracrine or Endocrine Signals in Liver Human hair growth defi ciency is linked to a genetic Development, Health, and Disease. Front. Cell. Dev. defect in the phospholipase gene LIPH. Science 314, Biol. 4, 30. 982-985. Jaks, V., Barker, N., Kasper, M., van Es, J.H., Snippert, Keyes, B.E., Segal, J.P., Heller, E., Lien, W.H., Chang, H.J., Clevers, H., and Toft gard, R. (2008). Lgr5 marks C.Y., Guo, X., Oristian, D.S., Zheng, D., and Fuchs, E. cycling, yet long-lived, hair follicle stem cells. Nat. (2013). Nfatc1 orchestrates aging in hair follicle stem Genet. 40, 1291-1299. cells. Proc. Natl. Acad. Sci. U. S. A. 110, E4950-9. Jamora, C., DasGupta, R., Kocieniewski, P., and Khatri, S.B., Edlund, R.K., and Groves, A.K. (2014). Fuchs, E. (2003). Links between signal transduction, Foxi3 is necessary for the induction of the chick otic transcription and adhesion in epithelial bud develop- placode in response to FGF signaling. Dev. Biol. 391, ment. Nature 422, 317-322. 158-169. Jussila, M., Aalto, A.J., Sanz Navarro, M., Shirokova, Khatri, S.B., and Groves, A.K. (2013). Expression of V., Balic, A., Kallonen, A., Ohyama, T., Groves, A.K., the Foxi2 and Foxi3 transcription factors during de- Mikkola, M.L., and Th esleff , I. (2015). Suppression of velopment of chicken sensory placodes and pharyn- epithelial diff erentiation by Foxi3 is essential for mo- geal arches. Gene Expr. Patterns 13, 38-42. lar crown patterning. Development 142, 3954-3963. Kobielak, K., Pasolli, H.A., Alonso, L., Polak, L., and Kadaja, M., Keyes, B.E., Lin, M., Pasolli, H.A., Fuchs, E. (2003). Defi ning BMP functions in the hair Genander, M., Polak, L., Stokes, N., Zheng, D., and follicle by conditional ablation of BMP receptor IA. J. Fuchs, E. (2014). SOX9: a stem cell transcriptional Cell Biol. 163, 609-623. regulator of secreted niche signaling factors. Genes Dev. 28, 328-341. Kobielak, K., Stokes, N., de la Cruz, J., Polak, L., and Fuchs, E. (2007). Loss of a quiescent niche but not fol- Kaestner, K.H. (2010). Th e FoxA factors in organo- licle stem cells in the absence of bone morphogenetic genesis and diff erentiation. Curr. Opin. Genet. Dev. protein signaling. Proc. Natl. Acad. Sci. U. S. A. 104, 20, 527-532. 10063-10068. Kaestner, K.H., Knochel, W., and Martinez, D.E. Kratochwil, K., Dull, M., Farinas, I., Galceran, J., and (2000). Unifi ed nomenclature for the winged helix/ Grosschedl, R. (1996). Lef1 expression is activated by forkhead transcription factors. Genes Dev. 14, 142- BMP-4 and regulates inductive tissue interactions in 146. tooth and hair development. Genes Dev. 10, 1382- Kalled, S.L., Ambrose, C., and Hsu, Y.M. (2005). Th e 1394. biochemistry and biology of BAFF, APRIL and their Kulessa, H., Turk, G., and Hogan, B.L. (2000). Inhibi- receptors. Curr. Dir. Autoimmun. 8, 206-242. tion of Bmp signaling aff ects growth and diff erentia- tion in the anagen hair follicle. Embo j. 19, 6664-6674.

56 References

Lalmansingh, A.S., Karmakar, S., Jin, Y., and Nagaich, Li, J., Zheng, H., Wang, J., Yu, F., Morris, R.J., Wang, A.K. (2012). Multiple modes of chromatin remodel- T.C., Huang, S., and Ai, W. (2012). Expression of ing by Forkhead box proteins. Biochim. Biophys. Acta Kruppel-like factor KLF4 in mouse hair follicle stem 1819, 707-715. cells contributes to cutaneous wound healing. PLoS One 7, e39663. Lam, E.W., Brosens, J.J., Gomes, A.R., and Koo, C.Y. (2013). Forkhead box proteins: tuning forks for tran- Li, N., Liu, S., Zhang, H.S., Deng, Z.L., Zhao, H.S., scriptional harmony. Nat. Rev. Cancer. 13, 482-495. Zhao, Q., Lei, X.H., Ning, L.N., Cao, Y.J., Wang, H.B., Liu, S., and Duan, E.K. (2016). Exogenous R-Spon- Laurikkala, J., Pispa, J., Jung, H.S., Nieminen, P., Mik- din1 Induces Precocious Telogen-to-Anagen Tran- kola, M., Wang, X., Saarialho-Kere, U., Galceran, J., sition in Mouse Hair Follicles. Int. J. Mol. Sci. 17, Grosschedl, R., and Th esleff , I. (2002). Regulation of 10.3390/ijms17040582. hair follicle development by the TNF signal ectodys- plasin and its receptor Edar. Development 129, 2541- Lien, W.H., and Fuchs, E. (2014). Wnt some lose some: 2553. transcriptional governance of stem cells by Wnt/be- ta-catenin signaling. Genes Dev. 28, 1517-1532. Lay, K., Kume, T., and Fuchs, E. (2016). FOXC1 main- tains the hair follicle stem cell niche and governs stem Lien, W.H., Polak, L., Lin, M., Lay, K., Zheng, D., and cell quiescence to preserve long-term tissue-regen- Fuchs, E. (2014). In vivo transcriptional governance erating potential. Proc. Natl. Acad. Sci. U. S. A. 113, of hair follicle stem cells by canonical Wnt regulators. E1506-15. Nat. Cell Biol. 16, 179-190. Lee, S.E., Sada, A., Zhang, M., McDermitt, D.J., Lu, Loizou, L., Andersen, K.G., and Betz, A.G. (2011). S.Y., Kemphues, K.J., and Tumbar, T. (2014). High Foxp3 interacts with c-Rel to mediate NF-kappaB re- Runx1 levels promote a reversible, more-diff erenti- pression. PLoS One 6, e18670. ated cell state in hair-follicle stem cells during quies- Lowry, W.E., Blanpain, C., Nowak, J.A., Guasch, G., cence. Cell. Rep. 6, 499-513. Lewis, L., and Fuchs, E. (2005). Defi ning the impact Lefebvre, S., and Mikkola, M.L. (2014). Ectodysplasin of beta-catenin/Tcf transactivation on epithelial stem research--where to next? Semin. Immunol. 26, 220- cells. Genes Dev. 19, 1596-1611. 228. Mardaryev, A.N., Meier, N., Poterlowicz, K., Sharov, Legue, E., and Nicolas, J.F. (2005). Hair follicle renew- A.A., Sharova, T.Y., Ahmed, M.I., Rapisarda, V., Lew- al: organization of stem cells in the matrix and the role is, C., Fessing, M.Y., Ruenger, T.M., et al. (2011). Lhx2 of stereotyped lineages and behaviors. Development diff erentially regulates Sox9, Tcf4 and Lgr5 in hair 132, 4143-4154. follicle stem cells to promote epidermal regeneration aft er injury. Development 138, 4843-4852. Legue, E., Sequeira, I., and Nicolas, J.F. (2010). Hair follicle renewal: authentic morphogenesis that de- Maretto, S., Cordenonsi, M., Dupont, S., Braghetta, P., pends on a complex progression of stem cell lineages. Broccoli, V., Hassan, A.B., Volpin, D., Bressan, G.M., Development 137, 569-577. and Piccolo, S. (2003). Mapping Wnt/beta-catenin signaling during mouse development and in colorec- Lehman, J.M., Laag, E., Michaud, E.J., and Yoder, B.K. tal tumors. Proc. Natl. Acad. Sci. U. S. A. 100, 3299- (2009). An essential role for dermal primary cilia in 3304. hair follicle morphogenesis. J. Invest. Dermatol. 129, 438-448. Matzuk, M.M., Kumar, T.R., Vassalli, A., Bickenbach, J.R., Roop, D.R., Jaenisch, R., and Bradley, A. (1995). Lemke, J.R., Kernland-Lang, K., Hortnagel, K., and Functional analysis of activins during mammalian de- Itin, P. (2014). Monogenic human skin disorders. velopment. Nature 374, 354-356. Dermatology 229, 55-64. McKoy, G., Protonotarios, N., Crosby, A., Tsatsopou- Levy, V., Lindon, C., Harfe, B.D., and Morgan, B.A. lou, A., Anastasakis, A., Coonar, A., Norman, M., (2005). Distinct stem cell populations regenerate the Baboonian, C., Jeff ery, S., and McKenna, W.J. (2000). follicle and interfollicular epidermis. Dev. Cell. 9, Identifi cation of a deletion in plakoglobin in arrhyth- 855-861. mogenic right ventricular cardiomyopathy with pal- Lewis, J.R., Reiter, A.M., Mauldin, E.A., and Casal, moplantar keratoderma and woolly hair (Naxos dis- M.L. (2010). Dental abnormalities associated with ease). Lancet 355, 2119-2124. X-linked hypohidrotic ectodermal dysplasia in dogs. Orthod. Craniofac. Res. 13, 40-47.

57 References

Mecklenburg, L., Paus, R., Halata, Z., Bechtold, L.S., Mustonen, T., Ilmonen, M., Pummila, M., Kangas, Fleckman, P., and Sundberg, J.P. (2004). FOXN1 is A.T., Laurikkala, J., Jaatinen, R., Pispa, J., Gaide, O., critical for onycholemmal terminal diff erentiation in Schneider, P., Th esleff , I., and Mikkola, M.L. (2004). nude (Foxn1) mice. J. Invest. Dermatol. 123, 1001- Ectodysplasin A1 promotes placodal cell fate during 1011. early morphogenesis of ectodermal appendages. De- velopment 131, 4907-4919. Mikkola, M.L., and Th esleff , I. (2003). Ectodysplasin signaling in development. Cytokine Growth Factor Myung, P.S., Takeo, M., Ito, M., and Atit, R.P. (2013). Rev. 14, 211-224. Epithelial Wnt ligand secretion is required for adult hair follicle growth and regeneration. J. Invest. Der- Mikkola, M.L. (2009). Molecular aspects of hypohi- matol. 133, 31-41. drotic ectodermal dysplasia. Am. J. Med. Genet. A. 149A, 2031-2036. Narhi, K., Jarvinen, E., Birchmeier, W., Taketo, M.M., Mikkola, M.L., and Th esleff , I. (2008). Sustained ep- Mikkola, M.L. (2008). TNF superfamily in skin ap- ithelial beta-catenin activity induces precocious hair pendage development. Cytokine Growth Factor Rev. development but disrupts hair follicle down-growth 19, 219-230. and hair shaft formation. Development 135, 1019- Mikkola, M.L. (2007). Genetic basis of skin append- 1028. age development. Semin. Cell Dev. Biol. 18, 225-236. Narhi, K., Tummers, M., Ahtiainen, L., Itoh, N., Th es- Mill, P., Mo, R., Fu, H., Grachtchouk, M., Kim, P.C., leff , I., and Mikkola, M.L. (2012). Sostdc1 defi nes the Dlugosz, A.A., and Hui, C.C. (2003). Sonic hedge- size and number of skin appendage placodes. Dev. hog-dependent activation of Gli2 is essential for Biol. 364, 149-161. embryonic hair follicle development. Genes Dev. 17, Nguyen, H., Rendl, M., and Fuchs, E. (2006). Tcf3 282-294. governs stem cell features and represses cell fate de- Morgan, B.A. (2014). Th e dermal papilla: an instruc- termination in skin. Cell 127, 171-183. tive niche for epithelial stem and progenitor cells in Niehrs, C. (2012). Th e complex world of WNT recep- development and regeneration of the hair follicle. tor signalling. Nat. Rev. Mol. Cell Biol. 13, 767-779. Cold Spring Harb Perspect. Med. 4, a015180. Nissen, R.M., Yan, J., Amsterdam, A., Hopkins, N., Morris, R.J., Liu, Y., Marles, L., Yang, Z., Trempus, C., and Burgess, S.M. (2003). Zebrafi sh foxi one modu- Li, S., Lin, J.S., Sawicki, J.A., and Cotsarelis, G. (2004). lates cellular responses to Fgf signaling required for Capturing and profi ling adult hair follicle stem cells. the integrity of ear and jaw patterning. Development Nat. Biotechnol. 22, 411-417. 130, 2543-2554. Motoyama, J., Heng, H., Crackower, M.A., Taka- Nowak, J.A., Polak, L., Pasolli, H.A., and Fuchs, E. batake, T., Takeshima, K., Tsui, L.C., and Hui, C. (2008). Hair follicle stem cells are specifi ed and func- (1998). Overlapping and non-overlapping Ptch2 tion in early skin morphogenesis. Cell. Stem Cell. 3, expression with Shh during mouse embryogenesis. 33-43. Mech. Dev. 78, 81-84. Nusse, R. (2005). Wnt signaling in disease and in de- Motoyama, J., Takabatake, T., Takeshima, K., and velopment. Cell Res. 15, 28-32. Hui, C. (1998). Ptch2, a second mouse Patched gene is co-expressed with Sonic hedgehog. Nat. Genet. 18, Ohyama, T., and Groves, A.K. (2004). Expression of 104-106. mouse Foxi class genes in early craniofacial develop- ment. Dev. Dyn. 231, 640-646. Mou, C., Jackson, B., Schneider, P., Overbeek, P.A., and Headon, D.J. (2006). Generation of the primary Ono, M., Yaguchi, H., Ohkura, N., Kitabayashi, I., hair follicle pattern. Proc. Natl. Acad. Sci. U. S. A. 103, Nagamura, Y., Nomura, T., Miyachi, Y., Tsukada, T., 9075-9080. and Sakaguchi, S. (2007). Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1. Na- Muller-Rover, S., Tokura, Y., Welker, P., Furukawa, ture 446, 685-689. F., Wakita, H., Takigawa, M., and Paus, R. (1999). E- and P-cadherin expression during murine hair follicle Ornitz, D.M., and Itoh, N. (2015). Th e Fibroblast morphogenesis and cycling. Exp. Dermatol. 8, 237- Growth Factor signaling pathway. Wiley Interdiscip. 246. Rev. Dev. Biol. 4, 215-266.

58 References

Oro, A.E., Higgins, K.M., Hu, Z., Bonifas, J.M., Ep- Plikus, M., Wang, W.P., Liu, J., Wang, X., Jiang, T.X., stein, E.H.,Jr, and Scott, M.P. (1997). Basal cell carci- and Chuong, C.M. (2004). Morpho-regulation of nomas in mice overexpressing sonic hedgehog. Sci- ectodermal organs: integument pathology and phe- ence 276, 817-821. notypic variations in K14-Noggin engineered mice through modulation of bone morphogenic protein Oshimori, N., and Fuchs, E. (2012). Th e harmonies pathway. Am. J. Pathol. 164, 1099-1114. played by TGF-beta in stem cell biology. Cell. Stem Cell. 11, 751-764. Plikus, M.V., Mayer, J.A., de la Cruz, D., Baker, R.E., Maini, P.K., Maxson, R., and Chuong, C.M. (2008). Osorio, K.M., Lee, S.E., McDermitt, D.J., Waghmare, Cyclic dermal BMP signalling regulates stem cell acti- S.K., Zhang, Y.V., Woo, H.N., and Tumbar, T. (2008). vation during hair regeneration. Nature 451, 340-344. Runx1 modulates developmental, but not injury-driv- en, hair follicle stem cell activation. Development 135, Plikus, M.V., Widelitz, R.B., Maxson, R., and Chuong, 1059-1068. C.M. (2009). Analyses of regenerative wave patterns in adult hair follicle populations reveal macro-envi- Osorio, K.M., Lilja, K.C., and Tumbar, T. (2011). ronmental regulation of stem cell activity. Int. J. Dev. Runx1 modulates adult hair follicle stem cell emer- Biol. 53, 857-868. gence and maintenance from distinct embryonic skin compartments. J. Cell Biol. 193, 235-250. Priller, M., Poschl, J., Abrao, L., von Bueren, A.O., Cho, Y.J., Rutkowski, S., Kretzschmar, H.A., and Ouspenskaia, T., Matos, I., Mertz, A.F., Fiore, V.F., and Schuller, U. (2011). Expression of FoxM1 is required Fuchs, E. (2016). WNT-SHH Antagonism Specifi es for the proliferation of medulloblastoma cells and in- and Expands Stem Cells prior to Niche Formation. dicates worse survival of patients. Clin. Cancer Res. Cell 164, 156-169. 17, 6791-6801. Page, M.E., Lombard, P., Ng, F., Gottgens, B., and Propper, A.Y., Howard, B.A., and Veltmaat, J.M. Jensen, K.B. (2013). Th e epidermis comprises autono- (2013). Prenatal morphogenesis of mammary glands mous compartments maintained by distinct stem cell in mouse and rabbit. J. Mammary Gland Biol. Neo- populations. Cell. Stem Cell. 13, 471-482. plasia 18, 93-104. Panteleyev, A.A., Botchkareva, N.V., Sundberg, J.P., Pummila, M., Fliniaux, I., Jaatinen, R., James, M.J., Christiano, A.M., and Paus, R. (1999). Th e role of the Laurikkala, J., Schneider, P., Th esleff , I., and Mikko- hairless (hr) gene in the regulation of hair follicle cata- la, M.L. (2007). Ectodysplasin has a dual role in ec- gen transformation. Am. J. Pathol. 155, 159-171. todermal organogenesis: inhibition of Bmp activity Paus, R., Muller-Rover, S., Van Der Veen, C., Maurer, and induction of Shh expression. Development 134, M., Eichmuller, S., Ling, G., Hofmann, U., Foitzik, K., 117-125. Mecklenburg, L., and Handjiski, B. (1999). A compre- Qiu, W., Li, X., Tang, H., Huang, A.S., Panteleyev, hensive guide for the recognition and classifi cation of A.A., Owens, D.M., and Su, G.H. (2011). Condition- distinct stages of hair follicle morphogenesis. J. Invest. al activin receptor type 1B (Acvr1b) knockout mice Dermatol. 113, 523-532. reveal hair loss abnormality. J. Invest. Dermatol. 131, Petiot, A., Conti, F.J., Grose, R., Revest, J.M., Hodiva- 1067-1076. la-Dilke, K.M., and Dickson, C. (2003). A crucial role Reddy, S., Andl, T., Bagasra, A., Lu, M.M., Epstein, for Fgfr2-IIIb signalling in epidermal development D.J., Morrisey, E.E., and Millar, S.E. (2001). Charac- and hair follicle patterning. Development 130, 5493- terization of Wnt gene expression in developing and 5501. postnatal hair follicles and identifi cation of Wnt5a as Phillips, D.J. (2000). Regulation of activin’s access to a target of Sonic hedgehog in hair follicle morphogen- the cell: why is mother nature such a control freak? esis. Mech. Dev. 107, 69-82. Bioessays 22, 689-696. Rendl, M., Lewis, L., and Fuchs, E. (2005). Molecular Pispa, J., Jung, H.S., Jernvall, J., Kettunen, P., Mus- dissection of mesenchymal-epithelial interactions in tonen, T., Tabata, M.J., Kere, J., and Th esleff , I. (1999). the hair follicle. PLoS Biol. 3, e331. Cusp patterning defect in Tabby mouse teeth and its Reya, T., and Clevers, H. (2005). Wnt signalling in partial rescue by FGF. Dev Biol. 216, 521-534. stem cells and cancer. Nature 434, 843-850. Rhee, H., Polak, L., and Fuchs, E. (2006). Lhx2 main- tains stem cell character in hair follicles. Science 312, 1946-1949.

59 References

Richardson, G.D., Bazzi, H., Fantauzzo, K.A., Waters, Schmidt-Ullrich, R., Tobin, D.J., Lenhard, D., Schnei- J.M., Crawford, H., Hynd, P., Christiano, A.M., and der, P., Paus, R., and Scheidereit, C. (2006). NF-kap- Jahoda, C.A. (2009). KGF and EGF signalling block paB transmits Eda A1/EdaR signalling to activate Shh hair follicle induction and promote interfollicular epi- and cyclin D1 expression, and controls post-initiation dermal fate in developing mouse skin. Development hair placode down growth. Development 133, 1045- 136, 2153-2164. 1057. Rimkus, T.K., Carpenter, R.L., Qasem, S., Chan, M., Schneider, M.R., Schmidt-Ullrich, R., and Paus, R. and Lo, H.W. (2016). Targeting the Sonic Hedge- (2009). Th e hair follicle as a dynamic miniorgan. hog Signaling Pathway: Review of Smoothened and Curr. Biol. 19, R132-42. GLI Inhibitors. Cancers (Basel) 8, 10.3390/can- Schutz, S., Fischer, U., Altvater, M., Nerurkar, P., cers8020022. Pena, C., Gerber, M., Chang, Y., Caesar, S., Schubert, Roberts, V.J., and Barth, S.L. (1994). Expression of O.T., Schlenstedt, G., and Panse, V.G. (2014). A messenger ribonucleic acids encoding the inhibin/ RanGTP-independent mechanism allows ribosomal activin system during mid- and late-gestation rat em- protein nuclear import for ribosome assembly. Elife bryogenesis. Endocrinology 134, 914-923. 3, e03473. Rompolas, P., and Greco, V. (2014). Stem cell dynam- Sennett, R., and Rendl, M. (2012). Mesenchymal-epi- ics in the hair follicle niche. Semin. Cell Dev. Biol. 25- thelial interactions during hair follicle morphogenesis 26, 34-42. and cycling. Semin. Cell Dev. Biol. 23, 917-927. Rompolas, P., Mesa, K.R., and Greco, V. (2013). Spa- Sennett, R., Wang, Z., Rezza, A., Grisanti, L., Roi- tial organization within a niche as a determinant of tershtein, N., Sicchio, C., Mok, K.W., Heitman, N.J., stem-cell fate. Nature 502, 513-518. Clavel, C., Ma’ayan, A., and Rendl, M. (2015). An Integrated Transcriptome Atlas of Embryonic Hair Rossi, A., Anzalone, A., Fortuna, M.C., Caro, G., Follicle Progenitors, Th eir Niche, and the Developing Garelli, V., Pranteda, G., and Carlesimo, M. (2016). Skin. Dev. Cell. 34, 577-591. Multi-therapies in androgenetic alopecia: review and clinical experiences. Dermatol. Th er. Shav-Tal, Y., and Zipori, D. (2002). Th e role of activin a in regulation of hemopoiesis. Stem Cells 20, 493- Sakakura, T., Kusano, I., Kusakabe, M., Inaguma, Y., 500. and Nishizuka, Y. (1987). Biology of mammary fat pad in fetal mouse: capacity to support development Shi, G., Sohn, K.C., Li, Z., Choi, D.K., Park, Y.M., of various fetal epithelia in vivo. Development 100, Kim, J.H., Fan, Y.M., Nam, Y.H., Kim, S., Im, M., et 421-430. al. (2013). Expression and functional role of Sox9 in human epidermal keratinocytes. PLoS One 8, e54355. Samuelov, L., Sprecher, E., and Paus, R. (2015). Th e role of P-cadherin in skin biology and skin patholo- Shimomura, Y., Wajid, M., Petukhova, L., Kurban, M., gy: lessons from the hair follicle. Cell Tissue Res. 360, and Christiano, A.M. (2010). Autosomal-dominant 761-771. woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair tex- Schepeler, T., Page, M.E., and Jensen, K.B. (2014). ture. Am. J. Hum. Genet. 86, 632-638. Heterogeneity and plasticity of epidermal stem cells. Development 141, 2559-2567. Snippert, H.J., Haegebarth, A., Kasper, M., Jaks, V., van Es, J.H., Barker, N., van de Wetering, M., van den Schlake, T. (2007). Determination of hair structure Born, M., Begthel, H., Vries, R.G., et al. (2010). Lgr6 and shape. Semin. Cell Dev. Biol. 18, 267-273. marks stem cells in the hair follicle that generate all Schmidt-Ullrich, R., Aebischer, T., Hulsken, J., Birch- cell lineages of the skin. Science 327, 1385-1389. meier, W., Klemm, U., and Scheidereit, C. (2001). Re- Sofaer, J.A. (1969). Aspects of the tabby-crin- quirement of NF-kappaB/Rel for the development of kled-downless syndrome. I. Th e development of tab- hair follicles and other epidermal appendices. Devel- by teeth. J. Embryol. Exp. Morphol. 22, 181-205. opment 128, 3843-3853. Solomon, K.S., Kudoh, T., Dawid, I.B., and Fritz, A. Schmidt-Ullrich, R., and Paus, R. (2005). Molecular (2003). Zebrafi sh mediates otic placode forma- principles of hair follicle induction and morphogene- tion and jaw development. Development 130, 929- sis. Bioessays 27, 247-261. 940.

60 References

Srivastava, A.K., Pispa, J., Hartung, A.J., Du, Y., Ezer, Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Low- S., Jenks, T., Shimada, T., Pekkanen, M., Mikkola, ry, W.E., Rendl, M., and Fuchs, E. (2004). Defi ning M.L., Ko, M.S., et al. (1997). Th e Tabby phenotype the epithelial stem cell niche in skin. Science 303, is caused by mutation in a mouse homologue of the 359-363. EDA gene that reveals novel mouse and human exons Vaahtokari, A., Aberg, T., Jernvall, J., Keranen, S., and and encodes a protein (ectodysplasin-A) with col- Th esleff , I. (1996). Th e enamel knot as a signaling lagenous domains. Proc. Natl. Acad. Sci. U. S. A. 94, center in the developing mouse tooth. Mech. Dev. 54, 13069-13074. 39-43. Stenn, K.S., and Paus, R. (2001). Controls of hair folli- Van Mater, D., Kolligs, F.T., Dlugosz, A.A., and cle cycling. Physiol. Rev. 81, 449-494. Fearon, E.R. (2003). Transient activation of beta -cat- Sternlicht, M.D., Kouros-Mehr, H., Lu, P., and Werb, enin signaling in cutaneous keratinocytes is suffi cient Z. (2006). Hormonal and local control of mammary to trigger the active growth phase of the hair cycle in branching morphogenesis. Diff erentiation 74, 365- mice. Genes Dev. 17, 1219-1224. 381. Van Mater, D., Kolligs, F.T., Dlugosz, A.A., and Fea- St-Jacques, B., Dassule, H.R., Karavanova, I., Botch- ron, E.R. (2003). Transient activation of beta -catenin karev, V.A., Li, J., Danielian, P.S., McMahon, J.A., signaling in cutaneous keratinocytes is suffi cient to Lewis, P.M., Paus, R., and McMahon, A.P. (1998). trigger the active growth phase of the hair cycle in Sonic hedgehog signaling is essential for hair devel- mice. Genes Dev. 17, 1219-1224. opment. Curr. Biol. 8, 1058-1068. Vidal, V.P., Chaboissier, M.C., Lutzkendorf, S., Cot- Suutari, M., Majaneva, M., Fewer, D.P., Voirin, B., sarelis, G., Mill, P., Hui, C.C., Ortonne, N., Ortonne, Aiello, A., Friedl, T., Chiarello, A.G., and Blomster, J. J.P., and Schedl, A. (2005). Sox9 is essential for outer (2010). Molecular evidence for a diverse green algal root sheath diff erentiation and the formation of the community growing in the hair of sloths and a specif- hair stem cell compartment. Curr. Biol. 15, 1340-1351. ic association with Trichophilus welckeri (Chlorophy- Visinoni, A.F., Lisboa-Costa, T., Pagnan, N.A., and ta, Ulvophyceae). BMC Evol. Biol. 10, 86-2148-10-86. Chautard-Freire-Maia, E.A. (2009). Ectodermal dys- Takagi, R., Ishimaru, J., Sugawara, A., Toyoshima, plasias: clinical and molecular review. Am. J. Med. K.E., Ishida, K., Ogawa, M., Sakakibara, K., Asakawa, Genet. A. 149A, 1980-2002. K., Kashiwakura, A., Oshima, M., et al. (2016). Bioen- Voutilainen, M., Lindfors, P.H., Trela, E., Lonnblad, gineering a 3D integumentary organ system from iPS D., Shirokova, V., Elo, T., Rysti, E., Schmidt-Ullrich, cells using an in vivo transplantation model. Sci. Adv. R., Schneider, P., and Mikkola, M.L. (2015). Ectodys- 2, e1500887. plasin/NF-kappaB Promotes Mammary Cell Fate via Takahashi, H., and Ikeda, T. (1996). Transcripts for Wnt/beta-catenin Pathway. PLoS Genet. 11, e1005676. two members of the transforming growth factor-beta Voutilainen, M., Lindfors, P.H., Trela, E., Lonnblad, superfamily BMP-3 and BMP-7 are expressed in de- D., Shirokova, V., Elo, T., Rysti, E., Schmidt-Ullrich, veloping rat embryos. Dev. Dyn. 207, 439-449. R., Schneider, P., and Mikkola, M.L. (2015). Ectodys- Tornqvist, G., Sandberg, A., Hagglund, A.C., and plasin/NF-kappaB Promotes Mammary Cell Fate via Carlsson, L. (2010). Cyclic expression of lhx2 regu- Wnt/beta-catenin Pathway. PLoS Genet. 11, e1005676. lates hair formation. PLoS Genet. 6, e1000904. Waghmare, S.K., Bansal, R., Lee, J., Zhang, Y.V., Mc- Trempus, C.S., Morris, R.J., Bortner, C.D., Cotsare- Dermitt, D.J., and Tumbar, T. (2008). Quantitative lis, G., Faircloth, R.S., Reece, J.M., and Tennant, R.W. proliferation dynamics and random chromosome (2003). Enrichment for living murine keratinocytes segregation of hair follicle stem cells. Embo j. 27, from the hair follicle bulge with the cell surface mark- 1309-1320. er CD34. J. Invest. Dermatol. 120, 501-511. Waluk, D., Zur, G., Kaufmann, R., Welle, M.M., Jagan- Tsai, S.Y., Sennett, R., Rezza, A., Clavel, C., Grisanti, nathan, V., Drogemuller, C., Muller, E.J., Leeb, T., and L., Zemla, R., Najam, S., and Rendl, M. (2014). Wnt/ Galichet, A. (2016). A Splice Defect in the EDA Gene beta-catenin signaling in dermal condensates is re- in Dogs with an X-Linked Hypohidrotic Ectodermal quired for hair follicle formation. Dev. Biol. 385, 179- Dysplasia (XLHED) Phenotype. G3 (Bethesda) 188. Wang, L., Siegenthaler, J.A., Dowell, R.D., and Yi, R. (2016). Foxc1 reinforces quiescence in self-renewing hair follicle stem cells. Science 351, 613-617.

61 References

Welle, M.M., and Wiener, D.J. (2016). Th e Hair Fol- Yan, M., Zhang, Z., Brady, J.R., Schilbach, S., Fair- licle: A Comparative Review of Canine Hair Follicle brother, W.J., and Dixit, V.M. (2002). Identifi cation of Anatomy and Physiology. Toxicol. Pathol. 44, 564- a novel death domain-containing adaptor molecule 574. for ectodysplasin-A receptor that is mutated in crin- kled mice. Curr. Biol. 12, 409-413. Wells, K.L., Hadad, Y., Ben-Avraham, D., Hillel, J., Cahaner, A., and Headon, D.J. (2012). Genome-wide Yang, J., Meyer, M., Muller, A.K., Bohm, F., Grose, SNP scan of pooled DNA reveals nonsense mutation R., Dauwalder, T., Verrey, F., Kopf, M., Partanen, J., in FGF20 in the scaleless line of featherless chickens. Bloch, W., Ornitz, D.M., and Werner, S. (2010). Fibro- BMC Genomics 13, 257-2164-13-257. blast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeo- Wiener, D.J., Gurtner, C., Panakova, L., Mausberg, stasis. J. Cell Biol. 188, 935-952. T.B., Muller, E.J., Drogemuller, C., Leeb, T., and Welle, M.M. (2013). Clinical and histological characteriza- Yang, J., Meyer, M., Muller, A.K., Bohm, F., Grose, tion of hair coat and glandular tissue of Chinese crest- R., Dauwalder, T., Verrey, F., Kopf, M., Partanen, J., ed dogs. Vet. Dermatol. 24, 274-e62. Bloch, W., Ornitz, D.M., and Werner, S. (2010). Fibro- blast growth factor receptors 1 and 2 in keratinocytes Wijchers, P.J., Hoekman, M.F., Burbach, J.P., and control the epidermal barrier and cutaneous homeo- Smidt, M.P. (2005). Cloning and analysis of the mu- stasis. J. Cell Biol. 188, 935-952. rine Foxi2 transcription factor. Biochim. Biophys. Acta 1731, 133-138. Zaret, K.S., and Carroll, J.S. (2011). Pioneer transcrip- tion factors: establishing competence for gene expres- Wilson, N., Hynd, P.I., and Powell, B.C. (1999). Th e sion. Genes Dev. 25, 2227-2241. role of BMP-2 and BMP-4 in follicle initiation and the murine hair cycle. Exp. Dermatol. 8, 367-368. Zhang, X., Ibrahimi, O.A., Olsen, S.K., Umemori, H., Mohammadi, M., and Ornitz, D.M. (2006). Receptor Wollenberg, A., and Eames, T. (2011). Skin diseases specifi city of the fi broblast growth factor family. Th e following a Christmas tree pattern. Clin. Dermatol. complete mammalian FGF family. J. Biol. Chem. 281, 29, 189-194. 15694-15700. Woo, W.M., Zhen, H.H., and Oro, A.E. (2012). Shh Zhang, Y., Tomann, P., Andl, T., Gallant, N.M., maintains dermal papilla identity and hair morpho- Huelsken, J., Jerchow, B., Birchmeier, W., Paus, R., genesis via a Noggin-Shh regulatory loop. Genes Dev. Piccolo, S., Mikkola, M.L., et al. (2009). Reciprocal 26, 1235-1246. requirements for EDA/EDAR/NF-kappaB and Wnt/ Xu, Z., Wang, W., Jiang, K., Yu, Z., Huang, H., Wang, beta-catenin signaling pathways in hair follicle induc- F., Zhou, B., and Chen, T. (2015). Embryonic atten- tion. Dev. Cell. 17, 49-61. uated Wnt/beta-catenin signaling defi nes niche loca- Zhang, Y.V., Cheong, J., Ciapurin, N., McDermitt, tion and long-term stem cell fate in hair follicle. Elife D.J., and Tumbar, T. (2009). Distinct self-renewal and 4, e10567. diff erentiation phases in the niche of infrequently di- Yamashiro, T., Aberg, T., Levanon, D., Groner, Y., and viding hair follicle stem cells. Cell. Stem Cell. 5, 267- Th esleff , I. (2002). Expression of Runx1, -2 and -3 278. during tooth, palate and craniofacial bone develop- Zhao, G.Q., and Hogan, B.L. (1996). Evidence that ment. Gene Expr. Patterns 2, 109-112. mouse Bmp8a (Op2) and Bmp8b are duplicated genes Yan, J., Xu, L., Crawford, G., Wang, Z., and Burgess, that play a role in spermatogenesis and placental de- S.M. (2006). Th e forkhead transcription factor FoxI1 velopment. Mech. Dev. 57, 159-168. remains bound to condensed mitotic and stably remodels chromatin structure. Mol. Cell. Biol. 26, 155-168.

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