Identification of the molecular

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Université Libre de Bruxelles Faculté de Médecine

I.R.I.B.H.M.

Identification of the molecular

mechanisms involved in the initiation, invasion and maintenance of basal cell

carcinoma

Jean-Christophe Larsimont

Thèse de Doctorat en Sciences biomédicales Promoteur : Professeur Cédric Blanpain

Année académique 2017-2018

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Remerciements

Je suis reconnaissant à tous les gens qui, de près ou de loin m’ont aidé durant ma thèse. Je m’excuse pour ceux qui ne sont pas inclus dans ces lignes mais le règlement veut que la section des remerciements ne soit pas plus volumineuse que la partie scientifique.

Je tiens d’abord à remercier mon promoteur, Cédric Blanpain, qui m’a accueilli au sein de son laboratoire et m’a permis de réaliser ma thèse tout en étant entouré gens brillants. C’est une personne très exigeante avec les gens qui composent son laboratoire mais il s’est toujours appliqué à lui-même un niveau d’exigence bien supérieur et sa brillante carrière en est le reflet.

Pas une fois je n’ai dû faire de concession dans le choix des expériences pour des raisons de financement ou parce que je ne pouvais pas collaborer avec des gens compétents. De par son travail acharné, il a toujours su réunir les meilleures conditions pour son équipe.

Son niveau d’exigence m’a aussi permis de dépasser de loin mes limites et de me développer bien plus en quelques années que je ne l’aurais fait en quelques décennies si je n’avais pas séjourné au sein de son laboratoire.

Dans un même registre, je tiens à remercier Khalil Kass Youssef, qui a eu la dure tâche de m’encadrer durant mes débuts. Il a été une personne clé dans mon apprentissage puisqu’il a su me diriger d’emblée dans la bonne direction en m’inculquant sa passion de la science et la rigueur avec laquelle il menait ses expériences. C’était également un soutien psychologique de premier plan grâce aux innombrables festivités qu’il organisait.

Autre personne clé durant ma thèse et dernière membre de la BCC team, Adriana. Aussi têtue que talentueuse, un simple regard à son historique de publication passé (et à venir) suffit à comprendre l’étendue de ses compétences et la passion qui l’anime. Ses compétences scientifiques n’ont d’égales que ses qualités humaines et plus qu’une amie, elle fait partie d’une sorte de famille, au même titre que Khalil, Adeline et Mélanie. Ces deux dernières sont également deux personnes aussi discrètes qu’admirables et sur lesquelles j’ai toujours pu compter. Je pense que le génotypage et toutes les tâches ingrates auraient pu être encore plus moroses sans elles.

Je tenais également à remercier le dernier membre de la famille BCC, Vijay, qui a toujours fait

3 preuve de bienveillance à mon égard et a toujours su faire preuve de bonne humeur malgré les moments difficiles qu’il a traversés.

A mon arrivée au labo, j’ai également pu compter sur un trio de rêve composé de Benjamin Beck, Benjamin Drogat et Gregory Driessens. Ben1 est probablement le scientifique le plus impressionnant que j’ai eu la chance de côtoyer. Véritable puit de savoir, je n’ai à ce jour pas encore trouvé un sujet qu’il ne connaisse pas et il semble maitriser toutes les techniques scientifiques existantes ainsi que l’ensemble des publications enregistrées sur Pubmed ces 60 dernières années. Si d’apparence il semble beaucoup plus « artistique », Gregory Driessens est tout aussi talentueux et m’a donné de précieux conseils durant ma thèse. Quant à papy Benjamin Drogat, si ses conseils scientifiques étaient précieux, c’est surtout la bonne humeur et la sérénité inébranlable qu’il incarnait qui m’ont été profitables.

Deuxième trio dont la deuxième position n’est que le reflet de l’ordre d’arrivée et non celui d’importance puisque je ne suis même pas certain que j’aurais pu terminer ma thèse sans eux.

En première position Souf, mon thesis buddy, avec qui j’ai cheminé depuis nos débuts d’étudiants célibataires, incapables de faire une extraction d’ARN en utilisant le kit le plus simple et le plus fiable du marché, jusqu’à sa publication dans le meilleur journal qui soit. Fort heureusement pour nous, nos vies privées ont également suivi la même tendance. Ce que je retiendrai de Souf, en plus d’un ami proche, c’est son aptitude à faire oublier à travers ses différents numéros quotidiens à quel point c’est un scientifique talentueux, et plus largement, un individu brillant.

Dans ce même trio, on retrouve Dany Nassar, la seule personne à avoir réussi à associer ces quatre trais a priori incompatibles : playboy, médecin, PhD et libanais. Il est également officieusement le meilleur brasseur du Liban. En dehors de son soutien au sein du labo et son amitié, je lui dois également le fait de m’avoir fait découvrir le monde de la bière tel que je le conçois aujourd’hui. Je pense que je me souviendrai à jamais de ce moment où il m’a fait découvrir la Punk IPA et que ma réponse fut « Dany, ce n’est pas une bière ça ».

On termine ce trio avec Fede qui bien qu’il soit arrivé après les autres, a rapidement su s’intégrer

à un point où on ne sait pas réellement retracer le moment où Fede est arrivé, c’est comme s’il

avait toujours été là. Italien au grand cœur, son calme et le recul qu’il arrive à prendre m’ont été

utiles à plus d’une fois. Bien qu’il soit foncièrement discret (en dehors de ses tirades d’injures en

4 italien envers les divers instruments du labo), c’est également une personnalité brillante comme en atteste les 942 langues qu’il parle couramment.

Troisième trio, Maud, Mathilde et Aline qui par leur humour bien à elles, ont rendu ces années plus légères qu’elles ne l’auraient été. Je retiendrai aussi particulièrement leurs performances tardives sur le dancefloor (et je pense que c’est largement réciproque).

Dernier trio, merci à Marielle, Sophie et Maria, non seulement pour avoir maintenu le niveau sonore de notre beau bureau et pour leur humour bien concon. Grâce à elles je peux me dire que j’étais dans « le bon bureau ». Merci à Guilhem d’avoir élevé le niveau de testostérone dans ce bureau rempli d’œstrogènes et également pour ses conseils scientifiques et son humour con.

Merci à Andrea, ma voisine, pour toutes les conversations, tant scientifiques que non- scientifiques et pour sa patience vis-à-vis de l’organisation un peu sommaire de mon bureau qui empiétait plus que largement sur son espace vital. J’espère qu’elle reviendra rapidement vers la science car elle est extrêmement talentueuse et a beaucoup à y apporter.

Merci à Gaëlle, aka maman, qui est probablement une des personnes ayant le plus contribué à l’achèvement de ma thèse tant elle abat un travail incroyable pour le labo et s’assure que tout aille bien pour tout le monde. Dans un même registre, merci à Farida de m’avoir rendu mon doigt, presque entier. Merci également à Nathy de s’être toujours coupée en 4 (si pas plus) pour toutes les démarches que j’ai pu avoir à effectuer.

Enfin, merci à toutes les techs du labo, Doriana, Christine, Gaëlle, Erwin, Catherine, Virginie pour leur boulot dont j’ai largement profité tout au long de mes travaux.

D’un point de vue scientifique et personnel, je tiens à remercier Véronique Del Marmol et Jean-

Marie Vanderwinden qui en plus d’être de grands scientifiques dont la force de la passion après

autant d’années (désolé) est admirable, sont deux personnes de très grande qualité sur le plan

humain et que je prends plaisir à revoir les trop rares fois où l’occasion se présente.

5 Merci aux membres du laboratoire de François Fuchs et en particulier à Mathieu Defrance pour le travail incroyable et central qu’il a réalisé dans mon projet. Merci à Pieter Baatsen pour sa guidance dans les limbes de la microscopie électronique.

Merci également à ma famille et en particulier, à ma mère et mon père qui venant de tout en bas ont réussi le pari de m’amener aussi loin que j’ai eu les capacités d’aller. Merci aussi d’avoir fait en sorte que je puisse faire tout ça sans avoir à me soucier de tout le reste.

Merci aussi à Thibaut et Laeti d’avoir été là jusqu’au bout, et par « au bout », j’entends jusqu’au déménagement chaotique du kot que j’avais ambitieusement entrepris seul. Merci également de m’avoir comblé en me permettant d’éventuellement obtenir le statut de Docteur Parrain.

Merci au reste de la famille, Mali, Françoise et en particulier à mamy de nous avoir élevés avec les valeurs qui sont les siennes, qu’on continue de porter et qui ont également rendu tout ça possible.

Ils ont également leur place dans la section famille, ceux avec qui j’ai partagé le logement pendant quelques mois voire années pour certains, Anne, Bérangère, Mathieu, Margot, Simon, Yannis. Ils ont tous un jour ou l’autre, indirectement contribué à la réussite de ce travail. Il en va de même pour les élus de l’ATM, Florent, Coco, William et Mika.

Enfin, merci à Maëlle pour son soutien et sa patience tout au long de ces travaux, des heures et

week-end passés à autre chose que vivre notre vie. Elle est d’ores et déjà responsable du fait

qu’obtenir ce doctorat ne sera jamais la meilleure chose qui me soit arrivée dans la vie.

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Table of contents

Table of abbreviations ... 8

1) Abstract ... 11

2) Résumé ... 12

3) Introduction ... 13

3A) Skin biology ... 13

3AA) Embryogenesis ... 14

3AAa) Epidermal specification ... 14

3AAb) Hair follicle embryogenesis ... 19

3AB) Homeostasis ... 20

3Aba) Stem cells ... 20

3AD) Skin cancers ... 30

3ADa) Basal cell carcinoma ... 31

3ADa2) Cell of origin ... 33

3B) Sox9 ... 38

3BA) Molecular characteristics ... 38

3Bb) SOX9 in embryogenesis ... 40

3Bc) Sox9 in epidermis ... 41

3Bd) SOX9 in cancer ... 44

3C) Resistance to therapy ... 47

3CBb) Mechanisms of resistance to therapy ... 48

3CBba) Drug efflux ... 48

3CBbb) Drug activation or inactivation ... 49

3CBbc) Alteration of drug targets ... 49

3CBbf) Redundancy ... 52

3CBbh) Cancer stem cells ... 54

3D) Resistance to therapy in BCC ... 56

4B) Lgr5 in adult tissues ... 59

4C) Lgr5 in cancer and cancer stem cells ... 60

5A) Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion 60 First paper: Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion... 65

5B. Mechanisms of BCC resistance to therapy ... 66

7 Second paper: A slow cycling Lgr5 tumor cell population mediates resistance to Smoothened inhibitor

in Basal Cell Carcinoma ... 69

6) Discussion ... 70

7. Perspectives. ... 81

References. ... 85

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Table of abbreviations

5FU : 5-fluoro-uracile ABC : ATP-binding cassette ACD : Asymmetric cell division AMH : anti-Müllerian hormone APC : Adenomatosis polyposis coli AR : Androgen receptor

BCC : basal cell carcinoma

BCRP : breast cancer resistance protein BM : basement membrane

BRAFI : BRAF inhibitors

CAF : cancer associated fibroblast CBC : carcinome basocellulaire CMD : campomelic dysplasia CP : committed progenitor DIM : dimerization domain DT : diphtheria toxin

DTA : diphtheria toxin fragment A DTR : diphtheria toxin receptor ECM : extra cellular matrix

EDC : electrodessication and curettage

9 EHFP : embryonic hair follicle progenitors

EMT : epithelial to mesenchymal transition ER : estrogen receptor

F-actin : filamentary actin

FAK : focal adhesion kinase signaling

FIB/SEM : Focus Ion Beam Scanning Electron Microscopy GFP : green fluorescent protein

GRN : gene regulatory network HFSC : hair follicle stem cell HMG : high mobility group Inv : involucrin

IR : ionizing radiations LRC : label retaining cells LRR : leucine-rich repeat MDR : multi-drug resistance

MEF : mouse embryonic fibroblasts MMS : Mohs micrographic surgery MTX : methotrexate

NBCCS : nevoid basal cell carcinoma syndrome NES : nuclear export sequence

NLS : nuclear localization signal

ORS : outer root sheet

10 PDT : photodynamic therapy

PML : promyelotic leukemia Pol η : DNA polymerase η ROS : reactive oxygen species SCD : symmetric cell division SHG : second harmonic generation Smoi : Smoothened inhibitor TA : transit amplifying cells TAM : tamoxifen

TEM : transmission electron microscopy TF : transcription factor

TLS : translesion DNA synthesis

TMECQ : 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin

TMZ : temozolomide

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1) Abstract

Basal cell carcinoma (BCC) is the most frequent cancer in human, responsible on its own for more cases than the cumulated incidence of all cancers summed together. Recent works which have analyzed the early steps of tumorigenesis have paved the way for the understanding of tumorigenesis from the oncogenic hit to fully formed tumors. They have highlighted a central role for genes involved in the regulation of the embryonic progenitors of hair follicle stem cells.

In this work, we have studied the role of the transcription factor Sox9, a gene known to be crucial for the specification of these progenitors and hair follicle stem cells. We have shown that Sox9 is required for the progression of pre-neoplastic lesions to fully developed basal cell carcinomas and for the maintenance of oncogene-expressing cells. Using state-of-the-art genome analysis, we have shown that Sox9 acts both as an activator and a repressor of gene expression. We have shown that it directly promotes self-renewal, invasion and quiescence in cancer cells.

In the second part of this work, we have uncovered the mechanisms of action of the Hedgehog

inhibitor Vismodegib on cancer cells. Indeed, using genetic mouse models, we have shown that

the inhibitor impedes the embryonic reprogramming that occurs during BCC formation and

promotes the differentiation of BCC cells towards interfollicular epidermis, infundibulum and

sebaceous gland fates depending on their cellular origin. In addition, we found that a subset of

BCC cells that express Lgr5 and display active Wnt signaling are more resistant to Vismodegib

treatment in mouse models, and found indication that it might be the case in human patients

too. In addition, we have shown that combined inhibition of Wnt signaling (genetic as well as

chemical) together with Vismodegib treatment alleviates this resistance and results in the

complete eradication of BCC cells.

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2) Résumé

Le carcinome basocellulaire (CBC) est le cancer le plus fréquent chez l’homme et est à lui seul responsable de plus de cas que l’incidence cumulée de tous les autres cancers réunis. Des travaux récents qui se sont intéressés aux étapes précoces de la tumorigenèse ont fourni un support précieux pour l’analyse du développement tumoral précoce. Ils ont mis en évidence le rôle central de gènes impliqués dans les progéniteurs embryonnaires des cellules souches du follicule pileux. Dans ce travail, nous avons étudié le rôle du facteur de transcription Sox9, un gène connu pour être crucial dans la spécification de ces progéniteurs et dans les cellules souches du follicule pileux. Nous avons montré que Sox9 est requis pour la progression des lésions paranéoplasiques vers les CBC et qu’il était également requis pour la maintenance des cellules cancéreuses. En utilisant des méthodes d’analyse génomique de pointe, nous avons montré que Sox9 agit tant comme un activateur que comme un répresseur de la transcription. Nous avons montré qu’il promeut directement le renouvellement cellulaire, l’invasion ainsi que la quiescence des cellules cancéreuses.

Dans la seconde partie de ce travail, nous avons découvert le mécanisme d’action de l’inhibiteur

d’Hedgehog Vismodegib sur les cellules cancéreuses. En utilisant des modèles génétiques de

souris, nous avons montré que l’inhibiteur empêche la reprogrammation embryonnaire qui a

normalement lieu durant la formation du CBC et qu’il promeut la différentiation des cellules

cancéreuses vers une identité interfolliculaire, d’infundibulum ou sébacée suivant l’origine de ces

cellules. Nous avons également observé qu’une population caractérisée par l’expression du gène

Lgr5 ainsi que par une activation de la voie de signalisation Wnt est plus résistante au Vismodegib

chez la souris et nous avons trouvé des indices suggérant qu’il pourrait en être de même chez

l’humain. Enfin, nous avons pu montrer que l’inhibition combinée des voies de signalisation Wnt

et Hedgehog annihile cette résistance et résulte en la disparition complète des CBCs.

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3) Introduction

3A) Skin biology

Skin is the largest organ of the human body and is composed of several compartments that cooperate to fulfill its wide variety of functions. As shown in Figure 1, it comprises the stratified epithelium called epidermis, the conjunctive tissue called dermis and the hypodermis, a richly vascularized structure mainly composed of adipose tissue (Blanpain and Fuchs, 2006).

- Hypodermis –

The hypodermis, the deepest layer of the skin, is a loose connective tissue that is richly vascularized and composed of adipose tissue, collagen, elastin and nerves (Driskell et al., 2014).

It has numerous functions, the first of which is acting as a link between the dermis and the underlying mobile structures such as muscles and tendons. In addition, its high content in adipocytes organized in lobules separated by connective tissue provides a reserve of energy (Vega et al., 2009) and an insulating layer that prevents excessive heat losses (Driskell et al., 2014).

- Dermis –

The dermis is a connective tissue located between the hypodermis and the epidermis. It is mainly

composed of collagen, elastin fibers, glycosaminoglycans, proteoglycans and glycoproteins (Watt

and Fujiwara, 2011). The composition and proportions of these various components change

depending on body location, but also differ between the upper layer of the dermis (papillary

dermis) and the lower region of the dermis (reticular dermis). The upper part is mainly composed

of loosely arranged collagen fibers and hosts blood vessels as well as Meissner’s corpuscles (Vega

et al., 2009; Watt and Fujiwara, 2011). The blood vessels present in the papillary dermis help

providing nutrients for the surrounding cells as well as regulating body temperature. Meissner’s

corpuscles are mechanoreceptors found in skin that are responsible for light touch and are

Figure 1 : skin structure

Schematic representation of human skin showing the organization of the three layers : epidermis, dermis and hypo- dermis. The epidermis is composed of a stratified epithelium, called interfolicular epidermis (IFE), which is located between the pilosebeceous units and the hair and appendages such as the sebaceous gland. The sweat gland regu- lates body temperature and the excretion of various compounds through the pores. The epidermis is anchored in a conjonctive layer called the dermis which contains different populations of cells involved in many different functions such as its maintenance and in immunity, as well as blood vessels and nerves. Dermis tops the hypodermis which is composed of adipocytes, conjonctive fibers, nerves and is richly vascularized. It confers a reserve of energy as well as an insulating layer that prevents excessive heat loss. Adapted from Ebling & Montagna, Britanica 2006.

pores

hair

sweat gland

nerve

hair follicle sebaceous gland

blood vessels epidermis

dermis

hypodermis

14 therefore found in greater abundance in areas that need precise and sensitive perception, such as fingers and lips. The reticular dermis is composed of a dense arrangement of collagen, elastic and reticular fibers that confer to the skin strength and resistance to stress (Watt and Fujiwara, 2011). In addition, the reticular dermis hosts various appendages such as nails, nerves, hairs, sebaceous glands as well as sweat glands. The dermis comprises various cell populations, including fibroblasts and melanocytes, which play a key role in skin function. Skin fibroblasts are a surprisingly heterogeneous population that greatly varies depending on body location (Driskell et al., 2013; Driskell and Watt, 2015; Lichtenberger et al., 2016). The dermal papilla fibroblasts are probably the best studied subpopulation and have crucial roles during skin embryogenesis, as well as in regulating hair follicle cycles (Driskell et al., 2013; Lichtenberger et al., 2016).

Skin melanocytes originate from the neural crest. They are responsible for producing melanin, the pigment responsible for skin pigmentation and that protects epidermal cells from UV induced damages (D'Mello et al., 2016).

- Epidermis –

The uppermost layer of the skin is a stratified epithelium called epidermis. It’s composed of hair follicles and their appendages (sweat glands, sebaceous glands) and the compartment located between hair follicles called interfollicular epidermis. It’s responsible for the protective function of skin against stress and pathogens, as well as for preventing water loss. The embryogenesis and homeostasis of the various compartments of the epidermis are covered in greater detail in sections 3A and 3B.

3AA) Embryogenesis

3AAa) Epidermal specification

The skin emerges from the ectoderm as a single layer of epidermal cells following inductive

signals from the mesenchymal cells at embryonic day 9.5 (E9.5). When mesenchymal cells start

populating the dermis around embryonic day 12.5 (E12.5), they secrete signals that instruct the

stratification of the skin epidermis (Ferraris et al., 2000). The nature of these signals is unclear,

15 but studies in chick embryo indicate a role of Wnt signaling (Wilson et al., 2001), while other studies performed in zebrafish and xenopus suggest that it relies on Bmp signaling (Hawley et al., 1995; Nikaido et al., 1999; Suzuki et al., 1997a; Suzuki et al., 1997b; Wilson and Hemmati- Brivanlou, 1995). These variations could be due to differences in the nature of the epidermis of these species, the two last being aquatic organisms, but it’s also possible that there is a cross-talk and/or redundant actions between the two pathways. Indeed, such interactions between Wnt and Bmp have been previously described in chicken skin (Wilson et al., 2001). One of the first players of epidermal stratification is the transcription factor p63 (Mills et al., 1999; Yang et al., 1999). Mice deficient for p63 are born with a single layer of ectodermal cells as a result of their absence of epidermal commitment (Koster and Roop, 2004; Wilson et al., 2001).

Around E14.5-15.5, this primitive single layered epithelium that was dividing parallel to the basal

lamina progressively switches its mode of division towards a perpendicular orientation,

generating the suprabasal spinous layer (Blanpain and Fuchs, 2006; Blanpain et al., 2007; Lechler

and Fuchs, 2005; Williams et al., 2011; Williams and Fuchs, 2013; Williams et al., 2014). The

spindle orientation can determine whether a division will be asymmetric (asymmetric cell

division, ACD) or symmetric (symmetric cell division, SCD). In the SCD, a basal cell gives rise to

two daughters with identical cell fate. In the ACD, a basal cell divides and gives rise to two

daughter cells with different cell fate (Knoblich, 2010). Note that while cell fate can be correlated

to spindle orientation, such as a perpendicular cell divisions that give rise to one basal and one

suprabasal cell (therefore being an ACD), or when a basal cell divides parallel to the basal lamina

and gives rise to two basal daughter cells (SCD), it is not necessarily depending on it. Indeed, the

vast majority of cell divisions in adult epidermis are found to be parallel to the basal lamina

(>70%), while lineage-tracing experiments predict that most of the basal cells divide in an

asymmetric manner (Clayton et al., 2007; Doupe et al., 2010; Mascre et al., 2012; Niessen et al.,

2013; Sanchez-Danes et al., 2016). Therefore, it appears that a significant part of parallel cell

divisions are ACDs where one of the daughter cells loses its adhesion and delaminates (Watt and

Green, 1982). The disruption of cell division orientation results in defects of epidermal

stratification (Lechler and Fuchs, 2005; Williams et al., 2011). However, the disruption of cell

orientation through knock-down of Lgn, mInsc or Gnai does not result in the complete absence

16 of stratification due to the existence of other mechanisms of stratification, such as delamination, that were shown to take place during skin embryogenesis (Williams et al., 2014).

As show in Figure 2, the axis of division can influence the segregation of fate polarity proteins and cell fate determinants, therefore regulating the ratio of ACD/SCD (Knoblich, 2010). In addition, a perpendicular axis of division can ensure that one daughter cell is eliminated from the basal lamina, therefore maintaining a constant number of basal cells. This highlights the importance of the cell division axis in the regulation of epidermal development and homeostasis.

During interphase and until the prophase, all cells have their centrosomes located at the apical pole. During prophase, some cells maintain them apically while others organize bipolar spindles in random orientations with no clear signs of what the division axis will be (Poulson and Lechler, 2010). The final orientation of the spindle is established during metaphase.

Recent studies have brought a significant knowledge about how the spindle can be connected to the polarity cues such as Par3, Par6, and aPKC (Figure 2b) (Williams et al., 2014). Gain- and loss- of-function experiments have highlighted the role of the protein Inscuteable (Insc) in organizing such a connection. Insc binds to the apically located Par3 protein, and in Par3-deficient neuroblasts Insc remains located in the cytoplasm, showing the importance of Par3 in the apical recruitment of Insc (Knoblich, 2010). Once located at the cell cortex, Insc recruits the adaptor protein Lgn that binds the heterotrimeric G protein subunit Gαi through its Goloco domains.

During interphase, Lgn is found in an inactive conformation, and it is the binding to Gαi that switches it to an active conformation and leads to its cortical recruitment (Poulson and Lechler, 2012). This ultimately leads to the recruitment of NuMA to the complex. Since NuMA directly interacts with the microtubules through its C-terminal tail, it appears clear that the Par3-Insc- Lgn-NuMA complex mediates the connection between the cell polarity apparatus and the mitotic spindle (Knoblich, 2010; Poulson and Lechler, 2012). Interestingly, Abl1-mediated phosphorylation of NuMA is required for its anchoring to the cell cortex, and Abl1 knock-out mice display random cell division axis distribution (Matsumura et al., 2012).

Importantly, several cues direct cell polarity and cell division axis in skin. Indeed, the knock-out

of the Srf gene results in mislocalized NuMA and Lgn as well as defects in spindle orientation,

Figure 2 : Mechanisms of asymmetric cell division.

(a) Schematic representation of the asymmetric segregation of the polarity proteins in the Drosophila neuroblast and its effect on the fate specification of the daughter cells : the cell receiving aPKC-Baz-Par6, Insc and Pins-Gai-Mud will become a neuroblast while the cell receiving Numb, Brat and Prospero will become a ganglion mother cell. (b) Scheme depicting symmetric and asymmetric cell division in the mammalian IFE. While divisions parallel to the basal lamina will result in symmetric segregation of the determinants Par3 and aPKC, perpendicu- lar cell division will result in the asymmetric segregation of these polarity markers and in the generation of one basal and one suprabasal cell. Adapted from Niessen et al. Journal of Cell Science 2012.

17 highlighting the importance of the actomyosin system in regulating spindle orientation (Luxenburg et al., 2011). Additionally, knock-out of α-catenin, another protein linked to the actin cytoskeleton, results in randomized location of Lgn and NuMA as well as cell division orientation, underscoring the importance of cues such as cell adhesion in regulating the cell division axis (Lechler and Fuchs, 2005). Similarly, knock-out of integrin-β1 results in randomized localization of Lgn and of the cell division axis (Lechler and Fuchs, 2005). Whether these are the result of the role of integrin-β1 and α-catenin in cell adhesion, cytoskeleton or both is unknown. In contrast, p63 knock-out keratinocytes lack apical polarity of Lgn and divide almost exclusively parallel to the basal lamina (Lechler and Fuchs, 2005). However, it’s unclear whether this is the result of a direct role of p63 in regulating spindle orientation or whether it’s the absence of proper epidermal specification that indirectly results in those defects.

While a lot of research has shown what regulates the perpendicular cell division that occurs during embryogenesis, it remains unclear how parallel cell division is driven. Of note, epidermal deletion of aPKCλ results in an increased rate of perpendicular cell divisions (Niessen et al., 2013).

The exact mechanism is unknown, and how aPKC can be important in directing both parallel and perpendicular cell divisions has not been resolved yet. One possible explanation is the presence of a second form of aPKC called aPKCζ, although mice deficient for aPKCζ do not display any obvious epidermal phenotype. Another putative mechanism that might induce parallel cell division could be the suppression of either Lgn or Insc and Gnai3, since their respective knock- down results in parallel cell division.

Several studies have focused on determining how the differential segregation of polarity markers can affect cell fate. Williams and colleagues have reported that knock-down of Lgn in vivo results in the absence of Notch activation in the suprabasal cells (Williams et al., 2011; Williams et al., 2014). Interestingly, they could fully rescue the defects generated by Lgn knock-down by overexpressing NICD to restore Notch activation.

Another hypothetical mechanism could arise from the fact that EGFR is asymmetrically

distributed in dividing human keratinocytes in vitro (Le Roy et al. 2010). Interestingly, the EGFR-

population is more quiescent than the EGFR+ population, and inhibition of EGFR induces the

expression of K1 and K10 (Peus et al., 1997). Therefore, the EGFR+ population may represent a

18 less differentiated stage of human keratinocytes with higher proliferation potential, while the EGFR- might be committed cells that exit the cell cycle and enter the terminal differentiation program.

Shortly after the formation of the periderm, a transient structure during epidermal development, the basal cells start producing the first suprabasal layer that is positive for K1 but is actively proliferating, two attributes that are found in suprabasal keratinocytes exclusively during embryogenesis. At E15.5, these intermediate suprabasal cells exit the cell cycle, differentiate in spinous cells that further differentiate into granular and cornified cells later during embryogenesis. The intermediate suprabasal cells also have the ability to directly differentiate into spinous, a process regulated by the ΔNp63 – Ikkα axis (Koster and Roop, 2007). This axis is involved in the production of K1 expressing cells, as well as in inducing their withdrawal from the cell cycle. As a result, Ikkα deficient mice display a larger K1 compartment because suprabasal cells fail to exit the cell cycle. Mice deficient for Irf6, 14-3-3σ and Ovol1 display a similar accumulation of intermediate cells (Koster and Roop, 2007).

Notch signaling has been shown to play a crucial role during the transition from basal to spinous cells (Blanpain et al., 2006). Conditional ablation of RBPJ, the transcription factor acting downstream of the Notch signaling cascade, results in complete absence of specification to spinous cell fate (Blanpain et al., 2006). Accordingly, the specification of spinous fate is impaired by conditional ablation of Hes1, a central Notch-target gene in skin epidermis (Moriyama et al., 2008). Mirroring these observations, Notch ectopic activation results in excessive commitment of basal cells to a spinous fate (Blanpain et al., 2006). Wang and colleagues have shown that Notch acts in part by regulating the expression of C/EBP DNA binding proteins, which cooperate with the AP2 transcription factors family to regulate terminal differentiation (Wang et al., 2008).

The transition from spinous to granular cells is regulated by a gradient of extracellular Ca

that

is established in utero. The gradient continuously increases from the basal layer to the cornified

envelope. The Ca

/PKC axis acts through PKCs and regulates the transition from the spinous to

granular cells where it downregulates the expression of K1/K10 and promotes the expression of

the markers of upper layers such as loricrin, filaggrin and transglutaminase (Bickenbach et al.,

1995; Yuspa et al., 1989). The extracellular calcium-sensing receptor (CasR) is an alternative mean

19 of Ca

to promote differentiation in a PKC-independent manner. Mice lacking CasR show reduced expression of filaggrin and loricrin and display abnormal keratohyalin granules and precocious lamellar body secretion. Mice overexpressing CasR mirror these phenotypes (Turksen and Troy, 2003).

The transition from granular cells to cornified envelope is regulated by the transcription factor Klf4. Mice deficient for Klf4 do not display obvious phenotypes in the basal and spinous layers.

However, they fail to form the mature cornified envelope, in part through the misregulation of Connexin26 and Sprr genes (Djalilian et al., 2006; Patel et al., 2003; Segre et al., 1999). Mice overexpressing Klf4 show accelerated barrier formation. In addition to Klf4, Grhl3 regulates barrier formation through the control of lipid metabolism and cell adhesion. Of note, Arnt and Gata3 deficient mice present epidermal envelope defects (de Guzman Strong et al., 2006; Geng et al., 2006) and a perturbation of lipid metabolism as well.

3AAb) Hair follicle embryogenesis

Along with the formation of the stratified epidermis, a second specification event takes place in order to form the hair follicle (HF) and its appendages. The first step takes place at E13/E14, and involves Wnt signaling mediated communication between the mesenchymal cells in the underlying dermis and the keratinocytes, which induces the condensation and fate reprogramming of a population of epidermal cells to form the hair placode (Blanpain and Fuchs, 2006). Simultaneously, secretion of Bmp-family ligands inhibits the placode fate in the adjacent cells. The mesenchymal cells remain closely located to the cells that have been instructed to form the future HF cells, and they will persist nearby the adult HF as a structure called dermal papilla.

The formation of the dermal papilla is dependent on Shh, Pdgfa, Recql4 and Sgk3 (Nakamura et

al., 2013). Its role in the adult HF homeostasis is detailed later. At this point, the placode shows

a strong proliferation, which leads to its elongation and progression towards the second stage of

HF morphogenesis, called hair peg. Shortly after that, distinct lineages become noticeable as

keratinocytes form the inner root sheath (IRS), followed by the generation of trichocytes that will

form the hair shaft. The IRS then become surrounded by a layer of K5/K14 expressing cells called

the outer root sheath (ORS). Interestingly, the first round of hair follicles generation does not

20 depend on HFSCs, although they’re specified early during embryogenesis (see section related to Sox9 function during embryogenesis for more details).

3AB) Homeostasis

3Aba) Stem cells

Tissues need to be maintained throughout the life of a living organism. This is ensured by homeostasis, a process defined as the ability of a cell or an organism to regulate its internal conditions in order to stabilize its health and functions, independently of the external conditions.

In many tissues, homeostasis is ensured by the presence of stem cells. Stem cells are cells that present an extended ability to self-renew and to differentiate into one or multiple lineages (Blanpain et al., 2007).

The classical model involves a hierarchy where the stem cell is located on top, and can generate differentiated cells that go towards the bottom of the pyramid in a unidirectional manner.

However, this model has recently been challenged by the ability of committed progenitors to dedifferentiate into stem cells, as well as by the presence of reserve cells that can replace stem cells upon ablation (Tata et al., 2013; Tian et al., 2011). These observations do not completely abrogate the validity of the previous model, but rather demonstrate that this hierarchical organization is much more plastic than previously thought.

Different stem cell populations are found in skin. They are presented in the following sections that cover the homeostasis of their respective compartment.

3ABb) The hair follicle and its stem cells

Adult hair follicle stem cells (HFSC) reside in a distinct anatomic location called the bulge. They

are responsible for the regeneration of the hair during the many cycles that hair will undergo

during life. The hair follicle cycle is depicted in Figure 3. At postnatal day 16, the hair follicle enters

a stage called catagen stage, when the matrix cells cease proliferation and the lower two-third of

the HF degenerate in a process involving apoptosis (Blanpain and Fuchs, 2006). The catagen is

Figure 3 : hair follicle cycle

After several cell cycles, matrix cells have exhausted their proliferative capacity and hair growth stops. This step is followed by the entry into a destructive phase (catagen), leading to the degeneration of the lower two-thirds of the follicle. The upper third of the follicle remains intact as a pocket of cells surrounding the old hair shaft (club hair) and the reservoir of hair follicle stem cells is located at the basis of this pocket. These cells are necessary to form a new hair follicle. After the catagen phase, the bulge cells enter a resting quiecent stage called telogen. At the end of the telogen rest, the hair follicles enter a new cycle of regeneration and hair growth (anagen stage). Adapted from Blanpain & Fuchs. Annual review ARCB -2006

Anagen II, stage IV

Anagen II, stage III

Anagen II, stage I Telogen I

Catagen II

Catagen I Anagen I

Bulge

Bulge

Bulge

Bulge

Hair germ Dermal

papilla

21 followed by a resting phase called telogen, where the dermal papilla is adjacent to the bulge due to the retraction that follows the degeneration of the lower HF. The resting phase is followed by the anagen phase, during which the hair regenerates thanks to the pool of HFSCs marked by CD34 expression (Blanpain et al., 2004; Tumbar et al., 2004). During this phase, the HFSCs generate transit amplifying cells located in the matrix, at the base of the follicle. These matrix cells have a high rate of proliferation and differentiate to produce the hair shaft and the inner root sheath.

While the HFSCs are multipotent and have the capacity to generate all lineages of the pilosebaceous unit, as well as repopulating the IFE upon wound healing, under normal conditions they do not contribute to the maintenance of IFE (Ito et al., 2005; Levy et al., 2005; Levy et al., 2007; Morris et al., 2004).

Additional populations of stem cells reside within the hair follicle, as show in Figure 4. Indeed, Jensen and colleagues have identified a subpopulation of cells that express Lrig1 and that are located in the junctional zone adjacent to the sebaceous gland and the infundibulum (Jensen et al., 2008). Under normal conditions, they contribute to the sebaceous gland as well as the interfollicular epidermis of the back skin. However, during skin reconstruction assays they can contribute to all skin lineages (Jensen et al., 2009; Watt and Jensen, 2009).

Moreover, a population of stem cells that is positive for Lgr6 is found at the junctional zone, and is committed to maintaining the junctional zone and the sebaceous gland (Snippert et al., 2010a).

Note that a population of cells that are negative for Lgr6 and positive for the antigen Mts24 (Plet1 gene) is found in the junctional zone above the Lgr6+ cells (Watt and Jensen, 2009). There is a significant overlap between Plet1 and Lrig1 expression, suggesting that these cells might be different states of a single population (Kretzschmar and Watt, 2014).

The sebaceous gland is maintained by a specific population of stem cells that express Blimp1 (Horsley et al., 2006). Of note, Jensen and colleagues have shown that Lrig1 expressing cells also participate to the homeostasis of the sebaceous gland (Jensen et al., 2009).

3ABc) Interfollicular epidermis and its stem cells

The interfollicular epidermis is a stratified epithelium located between hair follicles. It’s organized

Figure 4 : markers of epidermal stem cells.

Schematic representation of hair follicle in telogen phase showing the localization of the different stem cell popula-

tions identified in the infidibulum, the junctional zone, the isthmus, the sebaceous gland and the bulge. These popu-

lations are marked by Lrig1/Plet1, Lgr6, Blimp1 and Cd34 respectively. Adapted from Kretzschmar et al. CSH Perspec-

tive in Medicine 2014.

22 as a stack of basal, spinous, granular and cornified cells, from the lowest to the uppermost layer respectively (Blanpain and Fuchs, 2006). The basal layer is anchored to the basal membrane (BM), a 20-100µm thick layer of extracellular matrix that is produced by the epidermal cells with the help of dermal cells. The BM is composed of three layers: 1) the lamina lucida, that contains glycosaminoglycans including laminins; 2) the lamina densa, composed of type IV collagen, heparan sulfate and glycoproteins; 3) the lamina fibroreticularis, composed of fibronectin that hosts the anchorage fibrils (made of type VII collagen). Defects in several of these components, such as type VII collagen, cause severe skin diseases that display blistering of the epidermis (Bruckner-Tuderman et al., 1999). The basal layer of the epidermis is attached to the basal membrane through the interaction of its adhesion molecules (integrins), organized as macromolecular structures called hemidesmosomes with laminins that acts as a link with the underlying collagen fibers (Watt and Fujiwara, 2011). As hemidesmosomes are coupled with the keratins filaments through the internal plaque, they generate a strong tie between the conjunctive tissue and the epidermal cells.

The basal layer is the only proliferating layer of the skin composed of two distinct populations, a slow-cycling stem cell population and a more rapidly proliferating population of committed progenitors, with a hierarchical organization (Mascre et al., 2012). Mathematical modelling of clonal data has shown that both populations share a similar pattern of asymmetric self-renewal at the population level, where the balance between proliferation and differentiation is achieved through stochastic fate choices. Of note, these populations have different wound healing potential, with stem cells having the ability to strongly contribute to the long-term wound healing, while committed progenitors have a more restricted wound healing potential (Mascre et al., 2012).

Upon commitment to a differentiated stage, keratinocytes exit cell cycle, downregulate the

expression of basal adhesion molecules such as integrin α6 and β4 and move to the superior layer

called spinous layer (Blanpain and Fuchs, 2006). Spinous cells have strong villous cell-cell

connections that makes them particularly resistant to mechanic stresses. These connections are

mediated mainly through desmosomal adhesions. Desmosomes are a macromolecular complex

where each half of the attachment structure is located into one of two adjacent cells and clamp

23 their membranes together (Johnson et al., 2014). The junction between the two cells in the extracellular space is mediated by cadherins called desmoglein and desmocollin. These cadherins are anchored to the intracellular attachment plaque, which is composed of plakoglobin, plakophilin and desmoplakin (Johnson et al., 2014). Desmoplakin binds to the keratin filaments, and therefore joins the cell-cell junction with the intermediate filaments. The spinous cells then further differentiate into granular cells, which compose the last nucleated layer of the epidermis.

They present keratohyalin granules that will be cleaved into filaggrin in the upper layers of epidermis, as well as lipids that will be later secreted together with proteins as lamellar bodies forming a protective hydrophobic barrier.

The final stage of a keratinocyte journey is the differentiation into enucleated dead flat cells called squama that will be shed and replaced. This layer represents the first line of defense again external stress and pathogens. Its antipathogen action results from the presence of an acidic pH as well as antibacterial proteases.

This desquamation of corneocytes generates an imbalance in the tissue and must be compensated by new cells, a process called homeostasis. The existence of a population of stem cells in skin was first suggested after the observation that keratinocytes display three types of colonies when plated at clonal density in vitro: large colonies with a high proliferation potential (holoclones), composed of a majority of undifferentiated cells that comprise putative stem cells;

medium (meroclones) and small (paraclones) clones with little to no proliferative potential that are composed of cells in a more differentiated stage (Blanpain and Fuchs, 2006; Blanpain et al., 2007). These three types of clones have different propagation capacities, and holoclones keep the ability to self-renew and generate differentiated progeny, which are hallmarks of stem cells.

Using H³-thymidine label retaining experiments, Potten et al. have shown that the central cell of

the hexagonal column retains radioactive label much longer than the other cells, suggesting that

they have label-retaining properties, a feature previously thought to be a stem cell characteristic

(Loeffler et al., 1986; Potten, 1981; Potten et al., 1982). Based on these observation, they have

proposed a model called Epidermal Proliferative Unit (EPU) that contains a single stem cell at the

top of a cellular hierarchy and that maintains the whole column. This single stem cell will divide

asymmetrically, renewing itself and generating a cell with a more limited proliferation potential

24 called transit amplifying cells (TA). These TA cells will generate differentiated cells that will form the epidermal barrier. The TA will eventually exit the cell cycle and differentiate, being replaced by the asymmetric division of its columnar stem cell.

While very seducing due to its simplicity, several data challenge the validity of the EPU model.

Lineage tracing is a technique in which a cell is genetically labelled with a reporter that allows the monitoring of the cell and its progeny. Different strategies of lineage tracing using retroviruses and N-ethyl-N-nitrosourea to induce mutations that results in the expression of a fluorescent reporter have been performed and support the existence of EPU because they lead to the observation of columns of stained cells from the basal to the uppermost layer of the epidermis (Ghazizadeh and Taichman, 2001; Mackenzie, 1997; Ro and Rannala, 2005). However, these techniques present large biases. Indeed, retroviruses restrict the labelling exclusively to the proliferating cells. On the other hand, NENN is a well-known carcinogen, and its application on skin might modify the homeostatic behavior of the cells. To solve this last concern, the same study has been performed relying solely on the spontaneous activation of the reporter. However, it’s possible that the higher rate of mutation found in populations with a higher proliferating rate could lead to a preferential targeting of cells with a higher proliferation index, biasing the labelling. In addition, confocal analysis shows that clonally derived cells do not form restricted EPU structures.

These biases have been solved by the development of a new generation of transgenic mice that

allow the activation of a reporter without generating artificial stresses. This system is based on

the expression of a transgene encoding a CRE recombinase that recognizes and excises LoxP sites

when found as a pair, the combination of the two being known as the CRE/Lox system. The most

classic strategy using the CRE/Lox system consists of flanking a STOP cassette with two LoxP

sequences upstream of a reporter gene such as the green fluorescent protein (GFP). In addition,

the CRE transgene is placed downstream of a given promoter. The promoter can be lineage

specific (e.g. Klf4-CRE), region specific (e.g. Lgr5-CRE) or ubiquitous (e.g. Rosa26-CRE). The CRE

protein recognizes LoxP sites and induces deletion in between two LoxP sites in the same

orientation. The excision of the sequence will be transmitted to the progeny of the CRE

expressing cells. The sequence flanked by the two LoxP sites can be a gene of interest, and the

25 expression of the CRE therefore leads to the deletion of the gene (or some portion of it).

Alternatively, a genetic construct can be placed behind a STOP codon flanked by two LoxP sites.

This construct allows the expression of the genetic construct specifically in the CRE-expressing cells and their progeny.

Moreover, the CRE system can be temporally restricted, for example by fusing the CRE with the mutated binding domain of the human estrogen receptor (ER), that have the property to translocate into the nucleus (and therefore being active) solely upon the presence of the ER ligand Tamoxifen (TAM)(Feil et al., 1997). This allows the regulation of CRE activity in a timely manner, but also in a dose-dependent manner because a lower dose of TAM will target the CreER in less cells, allowing clonal labelling of cells.

Using this system, Doupe et al. and Clayton et al. have performed clonal lineage tracing in the skin epidermis and have demonstrated that basal clones present a very high differentiation rate, as shown by the fact that only 7% of basal clones remain after 3 months and only 3% after one year (Clayton et al., 2007; Doupe et al., 2010). In addition, the growth of clones is constant and never reaches a plateau, which also challenges the EPU theory. The authors performed mathematical modelling of the clonal data and proposed a model where basal cells are a hierarchy-free population of equipotent committed progenitors (CP) that have a stochastic probability of undergoing symmetric self-renewal (generating two CPs), asymmetric self-renewal (generating one CP and one differentiated cell) or symmetric differentiation (generating two differentiated cells). Based on the continuous growth of clones, the model also proposes the existence of a neutral drift, which results from the behavior of some clones that compensate for the rapid loss of others, a phenomenon observed in intestinal crypts (Snippert et al., 2010b).

However, the genetic mice model used by these studies presents a bias because they both use Ah-CREER, a model that has never been shown to be able to target the entire population of proliferating cells in the epidermis.

In order to solve these concerns, Mascré et al. have used two different models where the

expression of the CreER is driven by two skin-specific promoters: K14, that is expressed in the

entire basal layer of the epidermis, and Involucrin (Inv), that is expressed in suprabasal layers as

well as in a fraction of basal cells (Mascre et al., 2012). By using these two models to conduct

26 lineage tracing at the clonal level, they described that the basal cells expressing Inv have clonal dynamics characteristics that correspond to the committed progenitor population described by Clayton et al. The analysis of K14 clones reveals that they represent the stem cell population, which is more quiescent and gives rise to the Inv+ committed progenitors through stochastic fate decision. Further strengthening the fact that they represent different population with variations in cellular identities and properties, Mascré and colleagues have shown that while a few Inv+

cells participate to the wound healing process, they are not maintained at long term, quickly delaminate and are lost in squames. On the contrary, K14+ cells have a much higher capacity to be recruited to the wound and they are maintained at long term in the healed tissue.

However, this model fails to provide an explanation as to why two different types of epidermal differentiation, parakeratotic and orthokeratotic, coexist together in the tail epidermis.

Orthokeratotic differentiation corresponds to the standard description of epidermal differentiation, with the presence of a granular layer as well as absence of nuclei in the cornified layers. On the contrary, parakeratotic differentiation is characterized by the absence of a granular layer and the presence of nuclei in the cornified layers. In human, these two differentiation programs take place in different body locations. However, in mouse tail epidermis the region adjacent to hair follicles is called interscale and undergoes orthokeratotic differentiation, and surrounds regions called scale that undergo parakeratotic differentiation. By using a K14CREER system to perform lineage tracing at clonal doses, Gomez and colleagues (Gomez et al., 2013) have shown that the fraction of clones that cross the scale-interscale border is far lower than what should be observed if the epidermis was maintained as described by Mascré et al. On the contrary, clones seem to be restricted to the location where they arose from. The findings of Gomez et al. argue for a model in which the two IFE compartments would be maintained separately by unipotent stem cells.

Consistent with this hypothesis, Sada and colleagues have shown that there are at least two

different population of cells with different proliferation dynamics that are mostly restricted to

either the scale or the interscale region (Sada et al., 2016). Using fluorescently marked histone

H2B (H2B-GFP) that dilutes its signal after each division, the authors identified a population

located in the interscale region that has a low rate of division and therefore retained the H2B-

27 GFP labelling for a longer period of time. For this reason, they’re called label retaining cells (LRCs).

On the contrary, they found the non-LRCs to be localized in the scale region. Of note, the pattern of LRC and non-LRC location is also found in the back skin, even in the absence of the scale/interscale organization. However, their location seems to correlate with the location of blood vessels branch points that are preferentially localized in the dermis underlying non-LRCs.

When fitting the proliferation data on various model including the previous ones, the authors observed that the data fits well to a model in which there are two independent stem cells populations with different proliferation dynamics. Interestingly, Sada and colleagues profiled the transcriptome of LRCs and non-LRCs and identified Dlx1 and Slc1a3 as being upregulated in LRCs and in non-LRCs, respectively. Using these two cell-specific promoters as drivers of CREER expression, the authors performed lineage tracing. Interestingly, they discovered that Slc1a3 preferentially marked cells located in the scale area while Dlx1 was rather restricted to cells located in the interscale. Interestingly, not only these cell types were located in different and restricted locations, but so were their progeny. Finally, lineage ablation using the diphtheria toxin fragment A (DTA) has shown that the two populations are plastic during injury. However, only one of the two populations described shows a scaling behavior, a feature of stem cells, raising the possibility that a second stem cell population has not been found in that study. An alternative possibility comes from the work of Sánchez-Danés and colleagues, who have performed lineage tracing analysis at clonal doses using K14 and Inv promoters to drive the expression of the CREER, but this time also considering the location of the clones (Sanchez-Danes et al., 2016). By refining the analysis of these two cell populations, they have shown that two populations, a slow-cycling SC population and a rapidly dividing progenitor population, maintain the interscale region while the scale is maintained by a single CP population.

3AC) Signaling pathways regulating skin development and homeostasis.

Skin development and homeostasis are regulated by very complex signaling pathways that interact with each other and are tightly regulated in space and time (Blanpain and Fuchs, 2006).

The major signaling pathways involved in skin biology and relevant to this work are detailed

28 below.

3ACa) Hh signaling

Hedgehog owes its name to the striking phenotype observed in the mutant fruit fly Drosophila melanogaster that has been found at the beginning of the 20

century and displays a hair phenotype reminiscent of hedgehog spines (Briscoe and Therond, 2013; Ingham et al., 2011).

Hedgehog has three mammalian homologues named Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh). All Hh ligands are soluble proteins that undergo signal sequence cleavage and enter the secretory pathway during which they are cleaved, linked to a cholesterol molecule in their carboxy terminus before the adjunction of a palmitic acid group (Briscoe and Therond, 2013; Chen et al., 2004; Peters et al., 2004; Tukachinsky et al., 2012). At this point the cholesterol molecule prevents the Hh ligand from leaving the plasma membrane, and it needs the combined action of the transmembrane protein Disp (Dispatched) together with the secreted glycoprotein Scube2 (Tukachinsky et al., 2012).

As shown in Figure 5, once the Hh ligand has been released, it binds to its receptor Ptch (Patched)

and the co-receptors CAM-related/dowregulated by oncogenes (Cdo) and brother of Cdo (Boc),

as well as growth arrest-specific (Gas1). The co-receptors mediate the high affinity binding of Hh

multimers (Briscoe and Therond, 2013). The simultaneous absence of Gas1, Boc and Cdo

abolishes Hh activity. Binding of Ptch by the Hh ligand abolishes its inhibition on the GPCRs

protein Smo (Smoothened). The exact nature of the inhibition is unclear. Following the release

of Ptch inhibition, the inactive tail of Smo that rests in a closed conformation becomes

phosphorylated and adopts an open conformation. Smo can be phosphorylated successively by

protein kinase A (Pka), casein kinase 1α (Ck1α), Ck2 and Gpcr kinase 2 (Briscoe and Therond,

2013). Interestingly, the more Smo is phosphorylated, the stronger it activates the downstream

signaling. The cascade culminates with the Gli transcription factors, Gli1, Gli2 and Gli3 (Zhang et

al., 2011). When the pathway is in inactive state, Gli2 and Gli3 are phosphorylated by Pka, Ck1

and Gsk3β and subsequently cleaved into repressor forms called Gli2R and Gli3R. In the presence

of Hh ligand, Ptch is excluded from the primary cilium, Smo becomes phosphorylated as

Figure 5: HH signaling pathway

Schematic representation of the HH signaling pathway in «On» and «Off» states. In the absence of HH lignad, at the base of cilia, the GLI proteins GLI2 and GLI3 are phosphorylated by PKA, CKI and GSK3β. This leads to their proteoly- tic cleavage, leading to the generation of the repressor forms called GLI2R and GLI3R. In the presence of HH ligand, PTC and GPR161 exit the cilia, and SMO is phosphorylated by GPRK2 and CKI, and is gated into the primary cilium together with β-arrestin and the microtubule motor KIF3A. As a result, SMO, EVC (Ellis-van Creveld sundrome) and EVC2 become enriched in the cilium and SMO becomes active. The activation of SMO results in an increased cilia dwell time for SUFU and GLI2 and GLI3, the dissociation of the GLI–SUFU complex within the cilia and the transport of the activated full-length, GLI2 and GLI3 proteins from the cilia to the nucleus. There, GLIs will activate transcrip- tion. Adapted from Briscoe & Thérond Nat. Rev. Mol Cell Bio 10.1038/nrm3598

29 described above and enters the cilium together with β-arrestin and Kif3a (Briscoe and Therond, 2013). The activation and translocation of Smo to the cilia results in increased cilia dwell time for Sufu, Gli2 and Gli, as well as the dissociation of the Gli-Sufu complex. Gli2 and Gli3 in their full- length are then transported from the cilia to the nucleus in a process involving Kif7. Once present in the nucleus in their full-length form, they activate the transcription of their various target genes (Briscoe and Therond, 2013).

Hh signaling is essential for HF development and for the regulation of HF cycles. Shh null embryos develop hair placodes but fail to go past the hair bud stage (Chiang et al., 1999; St-Jacques et al., 1998; Wang et al., 2000). Interestingly, the importance of Hh signaling seems to be restricted to the epidermal cells, since transgenic expression of Gli2 in keratinocytes is sufficient to rescue the hair phenotype of Gli2 -/- mice. In adults, Shh is expressed in the matrix and in the developing germ, suggesting a role in hair regeneration. Consistent with this idea, the Hh inhibitor cyclopamine blocks anagen progression (Silva-Vargas et al., 2005). Conversely, activation of Hh signaling using Shh or small-molecule agonists accelerates the transition from telogen to anagen (Silva-Vargas et al., 2005).

While Shh plays a role in the hair cycle, Ihh is expressed in the sebaceous gland and is important for the growth and differentiation of sebocytes (Niemann et al., 2003).

3ACb) Wnt signaling

The biology of Wnt signaling keeps becoming more and more complex over the years. The pathway now includes more than 19 Wnt ligands as well as 15 receptors and co-receptors in seven different protein families (Anastas and Moon, 2013). Wnt signaling actually includes very different signaling processes such as canonical Wnt signaling, non-canonical Wnt-Ca² signaling as well as planar cell polarity (PCP). A comprehensive explanation of these different pathways is beyond the scope of this thesis and therefore the description will focus on canonical Wnt signaling because of its involvement in basal cell carcinoma pathogenesis.

Canonical Wnt signaling is characterized for being dependent on β-catenin for its signaling

cascade (Barker and Clevers, 2006; Papkoff et al., 1996). The pathway is depicted in Figure 6 and

Figure 6 : Wnt signaling pathway

In the absence of Wnt ligand, the excess of cytoplasmic β-catenin is targeted for degradation through

its association with a multiprotein complex. Upon binding of Wnt, its activated receptor complex recruits

certain key components of the β-catenin degradation targeting machinery. Stabilized free cytoplasmic

β-catenin is now translocated to the nucleus, where it can associate with transcription factors of the

LEF/TCF family to activate the expression of their target genes. Adapted from Blanpain & Fuchs

Annu. Rev. Cell Dev. Biol. 2006. 22:339–73

30 starts with the interaction of the Wnt molecule with its receptors Frizzled (Fzd), Lrp5 or Lrp6. The association of Wnt ligands with their receptor can be inhibited by a variety of inhibitors such as Wif (Wnt inhibitory factor), sFRP (secreted frizzled related protein), Dkk (Dickkopf-related protein) and Sost (Slcerostin). The association of Wnt with its receptors results in the inhibition of the glycogene synthase kinase 3 (Gsk3) that forms a complex with Axin, adenomatosis polyposis coli (APC) and casein kinase 1α (Ck1α), which normally phosphorylate and target β- catenin for ubiquitylation and proteasomal degradation. Following the interaction of Wnt with their receptor, Dvl is activated and inhibits the destruction complex that is targeting β-catenin for degradation, resulting in the accumulation of β-catenin. The stabilization of cytoplasmic β- catenin leads to its entry into the nucleus where it associates with transcription factors of the high mobility group (HMG) family, mostly Tcf7 (Tcf1), Tcf7l1 (Tcf3), Tcf7l2 (Tcf4) and Lef1 (lymphoid enhancer-binding factor), to regulate gene transcription.

Wnt signaling is the earliest molecular signal that is required to program epithelial cells into a HF fate. β-catenin mutant mice as well as mice overexpressing the Wnt inhibitor Dkk1 fail to form hair placodes (Andl et al., 2002; Huelsken et al., 2001) . Conversely, the expression of a constitutively active form of β-catenin leads to the formation of de novo hair follicles in ectopic locations such as IFE, SG and ORS (Gat et al., 1998).

In addition to its role during development, Wnt signaling also regulates the activation of HFSCs during the hair cycle. During the transition from resting to growing phase, HFSCs accumulate nuclear β-catenin, suggesting a role in this transition, although forced expression of stabilized β- catenin alone is not sufficient to activate bulge SCs (DasGupta and Fuchs, 1999; Lo Celso et al., 2004; Lowry et al., 2005). In addition, it’s also required for the differentiation of matrix cells to produce the hair shaft (Merrill et al., 2001).

3AD) Skin cancers

Skin cancers are a very heterogeneous group of tumors that can be subdivided into two

categories: non-melanoma skin cancers (including basal cell carcinoma, squamous cell carcinoma

and other neoplasms of the skin), which have by far the higher incidence but also the best

prognosis, and melanoma, which is more rare but deadlier.