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The in vivo function of mammalian cell and tissuepolarity regulators – how to shape and maintain theepidermal barrier

Michaela T. Niessen1,2, Sandra Iden3 and Carien M. Niessen1,3,*1Department of Dermatology, Center for Molecular Medicine, Robert Kochstrasse 21, 50931 Cologne, Germany2International Graduate School for Genetics and Functional Genomics (IGS-GFG), Graduate School for Biological Sciences, Institute for Genetics,Zulpicher Strasse 47a, 50674 Cologne, Germany3Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Institute for Genetics, Zulpicher Strasse 47a,50674 Cologne, Germany

*Author for correspondence (Carien.niessen@uni-koeln.de)

Journal of Cell Science 125, 3501–3510� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.092890

SummaryThe establishment and maintenance of cell and tissue polarity is crucial for a range of biological processes, such as oriented division,migration, adhesion and barrier function. The molecular pathways that regulate cell and tissue polarity have been extensively studied inlower organisms as well as in mammalian cell culture. By contrast, relatively little is still known about how polarization regulates the in

vivo formation and homeostasis of mammalian tissues. Several recent papers have identified crucial roles for mammalian polarityproteins in a range of in vivo processes, including stem cell behavior, cell fate determination, junction formation and maintenance andorgan development. Using the epidermis of the skin as a model system, this Commentary aims to discuss the in vivo significance of cell

and tissue polarity in the regulation of mammalian tissue morphogenesis, homeostasis and disease. Specifically, we discuss themechanisms by which the molecular players previously identified to determine polarity in vitro and/or in lower organisms regulateepidermal stratification; orient cell division to drive cell fate determination within the epidermal lineage; and orient hair follicles. Wealso describe how altered polarity signaling contributes to skin cancer.

Key words: Polarity, Cytoskeleton, Signaling, Adhesion, Epidermis, Hair follicle, Barrier function, Cancer

IntroductionPolarity is defined as the unequal distribution of molecules

(RNAs, lipids, proteins) within a cell to produce asymmetry in its

structure and function at the cellular, tissue and organismal level.

Such asymmetry is a basic feature of almost all cells, and is

important for a wide range of processes, including cell fate

determination, adhesion, migration, differentiation and stem cell

maintenance (reviewed by Nelson, 2003; St Johnston and

Ahringer, 2010). Polarity comes in two main forms: cell

polarity and tissue polarity. In cell polarity, asymmetry is

achieved in individual cells, such as for instance the leading and

trailing edge of a migrating cell or apicobasal polarity (Fig. 1A).

In tissue polarity, also known as planar cell polarity (PCP),

subcellular structures and/or cells are aligned in the plane of the

tissue (reviewed by Bayly and Axelrod, 2011; Klein and

Mlodzik, 2005), as seen, for example, in the positioning of

actin-based hair in Drosophila wing cells (Fig. 2A). Although it

was initially mainly studied in lower organisms or in epithelial

cell culture systems, it is now clear that polarization is a

fundamental requirement for the proper functioning of cells and

tissues and that inappropriate function of polarity pathways

disturbs tissue homeostasis, leading to a variety of human

diseases, such as kidney disease, hearing impairment and cancer

(Bulgakova and Knust, 2009; Huang and Muthuswamy, 2010;

Lee and Vasioukhin, 2008; McNeill, 2010).

In this Commentary, we will briefly introduce the major

molecular mediators that are important to establish cell and tissue

polarity and then discuss how different types of polarity are

established in a stratifying epithelium, the epidermis of the skin,

and which molecular players have been implicated in the

regulation of these different processes.

Core molecular regulators of polarityPolarity is established and maintained by the activity of a core set

of polarity proteins, most of which were first identified in

Drosophila melanogaster or Caenorhabditis elegans, and are

highly conserved throughout metazoa. The basic molecular

players that determine either cell or tissue polarity are not

identical. Cell polarity is regulated by a core set of proteins and

protein complexes (Table 1). The Par (partitioning-defective)

proteins were the first cell polarity proteins to be identified based

on their role in asymmetric cell division of the C. elegans zygote

(Kemphues et al., 1988). The PAR genes encode either adaptor

proteins, such as the PDZ-domain-containing proteins Par3 and

Par6 or the 14-3-3 protein Par5, or serine and threonine kinases

such as Par1 and Par4. Par3 and Par6 form a dynamic complex

with atypical protein kinase C (aPKC) (Suzuki and Ohno, 2006;

McCaffrey and Macara, 2009). Two additional cell polarity

complexes regulate a range of asymmetric cell processes: the

transmembrane protein Crumbs forms a complex with the PDZ

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Fig. 1. Functional similarities and differences of polarity in simple epithelia and stratifying epithelia. At present, it is unclear whether the same molecular

mechanisms controlling apico-basolateral polarity in simple epithelia are involved in the establishment of epidermal polarity. (A) apico-basolateral polarity in

simple epithelia. The apical junctional complex, consisting of tight junctions (TJs), adherens junctions (AJs) and desmosomes (DSs) forms a border to establish

apico-basolateral polarity. Mutual interactions between polarity proteins and complexes regulate apico-basolateral polarity and barrier formation in simple

epithelia. (B) Polarity in the mouse epidermis. In contrast to simple epithelia, the epidermis has no distinct apical and basolateral membrane domains, but displays

apico-basolateral polarity over the multilayered tissue with the stratum granulosum forming the viable apical border. This is reflected in differential expression or

localization of polarity proteins, for example PAR3, PAR6, LGL1, Crumbs and aPKC (black lines indicate localization patterns in the different layers of the skin),

and adhesive junctions. Moreover, lamellar bodies (blue circles) and keratohyalin granules (purple asterisks) are targeted (indicated by arrows) towards the upper

layers to form the cornified envelope. Par, partitioning defective; Dlg, discs large; Lgl, lethal giant larvae; aPKC, atypical protein kinase C; Patj, protein-

associated with tight junction; Pals1, proteins associated with Lin Seven-1.

Stratum corneumStratum granulosumStratum spinosumStratum basale

Sebaceousgland

Hairshaft

Wild type

Vangl2 mutant, Celsr1 mutant

Anterior Posterior

45°

D

Fig. 2. Mechanisms and mediators of planar cell polarity in the

Drosophila wing and mouse epidermis. (A) Single actin-based hairs

in wing cells of Drosophila polarize to localize at the proximal side in

all cells of the tissue (shown on the left). On the right, a schematical

top view on Drosophila wing cells at pupal stages is shown

illustrating the asymmetric distribution of core PCP proteins Pk–

Vang–Fmi group (proximal) and the Dsh–Dgo–Fz–Fmi group

(distal). (B) Polarized distribution of PCP proteins mediate

orientation of developing mouse hair follicles. In the embryonic

epidermal basal layer, E-cadherin is expressed at all cell–cell contact

sites, whereas CELSR1, VANGL2 and FZ are exclusively localized

at anterior and/or posterior lateral membranes already at placode

stage, whereas the budding hair follicle does not reveal any obvious

asymmetry. At hair germ stage, PCP signaling determines anterior

constriction of cells resulting in asymmetry, characterized within the

hair germ by anterior localized ZO-1 and P-cadherin and posterior

enrichment of NCAM and E-cadherin. (C) Orientation of hairs on

dorsal skin and paws of control Fz6+/2 and Fz62/2 (Frizzled-6

knockout) mice. Loss of FZ6 interferes with normal hair patterning

and leads to random waves, swirls and whorls in back skin (top panel)

and paws of mutant mice (bottom panel). Image kindly provided by

Jeremy Nathans. (D) Orientation of hair follicles in the skin. In dorsal

skin of newborn mice, hair grows uniformly in a posterior direction

and hair follicle angles encompass a range of roughly 6 45˚ relative

to the anterior–posterior (A-P) axis (left). In mice with mutations in

Vangl2 or Celsr1, this planar orientation of hair follicles is lost

(right). Pk, Prickle; Vang, Van Gogh; Fmi, Flamingo; Dsh,

Dishevelled; Dgo, Diego; Fz, Frizzled; Celsr1, cadherin EGF-like,

LAG-like, seven pass receptor (murine homolog of Drosophila

flamingo); VANGL2, murine homolog of Van Gogh; ZO-1, zona

occludens-1; NCAM, Neural cell adhesion molecule.

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adaptor proteins Patj (protein-associated with tight junction) and

Lin7, as well as the membrane-associated guanylate kinase

(MAGUK) scaffold Pals1 (proteins associated with Lin7)

(Assemat et al., 2008; Bulgakova and Knust, 2009). The other

complex consists of the leucine-rich repeat and PDZ (LAP)

protein Scribble, the WD40 repeat-containing protein Lgl (lethal

giant larvae) and the MAGUK protein Dlg (discs large) (Bilder,

2004; Humbert et al., 2008).

To establish and maintain cell polarity, these cell polarity

proteins function in an integrated network that controls their

subcellular localization through multiple positive- and negative-

feedback loops that are tightly regulated in a spatiotemporal

manner (Fig. 1A). This network interacts with small GTPases,

adhesive junctions, the cytoskeleton and vesicle transport to

regulate the establishment and maintenance of cell polarity

(Iden and Collard, 2008; Nelson, 2003). Although the basic

mechanisms are similar, each type of cell polarity requires the

engagement of different cell polarity proteins.

Tissue polarity is regulated by planar polarity proteins, which

engage in signal transduction to coordinate changes in cell shape

and polarity across cells in the plane of the tissue (McNeill, 2010;

Simons and Mlodzik, 2008). Currently, two main core PCP

pathways have been described, the Frizzled (Fz)–Dishevelled

(Dsh)–Van Gogh (Vang) pathway and the Fat–Dachsous (Ds)–

Four-joint (Fj) pathway, and their functions have been best

characterized in Drosophila (McNeill, 2010; Simons and

Mlodzik, 2008). Fz–Dsh–Vang proteins form asymmetric

domains within cells in the plane of the tissue (Fig. 2A),

whereas the Fat–Ds–Fj pathway establishes gradients over a

range of cells (Table 2). Because tissue polarity requires the

polarization of individual cells, the different polarity signal

pathways are integrated to coordinate cell with tissue polarity

(Simons and Mlodzik, 2008). For example, the cell polarity

protein Scribble genetically interacts with the PCP protein vang-

like 2 (VANGL2) in mice (Montcouquiol et al., 2003), and loss

of Scribble results in classical PCP phenotypes, such as neural

tube closure defects and impaired embryonic wound healing

(Caddy et al., 2010; Dow et al., 2007; Murdoch et al., 2003). For

an in-depth discussion of the function and interactions of core

polarity proteins, we refer the reader to several excellent reviews

that cover different aspects of polarity (Assemat et al., 2008;

Bilder, 2004; Bulgakova and Knust, 2009; Goldstein and Macara,

2007; Humbert et al., 2008; Iden and Collard, 2008; Knoblich,

2008; Li and Bowerman, 2010; Martin-Belmonte and Mostov,

2008; McNeill, 2010; Nelson, 2003; Shin et al., 2006; Simons

and Mlodzik, 2008; St Johnston and Ahringer, 2010).

Whereas in lower organisms most of the core polarity proteins

are unique, in mammals, several orthologs exist for nearly all

core polarity proteins (Tables 1, 2) (for references, see reviews

above). Redundancy or compensation might thus obscure the in

vivo relevance of a particular polarity protein when mouse

knockout models of these proteins are examined. Nevertheless,

the complete inactivation of several polarity proteins has already

revealed their conserved role in mammalian polarity-dependent

processes, such as FAT4, whose inactivation disturbs oriented

cell division and tubule elongation, resulting in cystic kidney

disease (Saburi et al., 2008).

The skin as an in vivo model system for polarity-driven processesMammalian skin protects the organism from dehydration and

provides a barrier against harmful influences, such as UV,

temperature and microbes. The epidermis forms the outermost

layer and consists of the interfollicular epidermis (IFE) and

Table 1. Cell polarity proteinsa

Original symbol Mammalian symbol Function

Par1b MARK2 Ser/Thr kinaseMARK1, MARK3, MARK4, MARK5?

Par3b PAR3A (ASIP) PDZ-containing scaffoldPAR3B

Par4b LKB1 Ser/Thr kinasePar5b 14-3-3 family (7 members, b, c, e, f, t, s, g) PhosphoSer/Thr binding scaffoldPar6b PAR6A

PAR6B PDZ-containing scaffoldPAR6C

aPKC (atypical protein kinase C)d aPKCl (m), aPKCi (h) Ser/Thr kinaseaPKCf

Lgl (Lethal giant larvae)c LGL1 (Hugl1) WD40-repeat-containing scaffoldLGL2

Scribblec Scribble (Vartul, Crib1, LAP4) LAP protein or scaffoldDlg (Discs Large)c DLG1 (Sap97), DLG2 (PSD-93), DLG3 (SAP-102),

DLG4 (PSD95, SAP90), DLG5MAGUK protein or scaffold

Crb (Crumbs)c CRB1 (RP12, LCA8), CRB2, CRB3 TM protein or scaffoldStd (Stardust)c PALS1 (MMP5), PALS2 (MMP6, VAM-1, p55T)

MMP1–MMP3, MMP7MAGUK protein or scaffold

Patj (Drosophila Pals1-associatedtight junction protein)c

PATJ (INADL), MUP1 PDZ-containing scaffold

Lin7b LIN7A (Velis1, MALS-1), LIN7B (Velis2, MALS-2),LIN7C (Velis3, MALS-3)

PDZ-containing scaffold

aOriginal symbols of polarity proteins, the organism in which they were first identified, symbols of the mammalian orthologs and known structural function.bIdentified in C. elegans.cIdentified in Drosophila.dIdentified in mammals.N.D., not determined; TM, transmembrane protein; h, human; m, mouse.

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epidermal appendages, such as the hair follicles, and sebaceous

and sweat glands (Koster, 2009). Epidermal keratinocytes

balance life-long self-renewal in the proliferating basal layer

with a strictly spatiotemporally regulated terminal differentiation

program that is necessary to form the stratum corneum, a dead,

cornified and water-impermeable cell layer (Koster, 2009).

Different populations of stem and progenitor cells that are

located in the basal layer of the IFE and in specific areas of hair

follicles guarantee constant self-renewal under steady state

conditions and ensure sufficient plasticity for the fast

replacement of lost tissue in case of injury (Blanpain and

Fuchs, 2009; Watt and Jensen, 2009).

The epidermis is therefore an excellent cell biology model

system to study a range of processes that require polarization,

such as (1) formation and maintenance of a life-long self-

renewing stratifying epithelium and its appendages; (2) the

behavior of different populations of stem or progenitor cells that

drive epidermal self-renewal and cyclic regeneration of the hair

follicle; (3) coupling of cell fate decisions to oriented cell

division; (4) the highly organized spatial distribution of hair

follicles and sebaceous glands; and (5) wound healing as a model

for migration and tissue regeneration. Another advantage of this

model system is that primary keratinoyctes can be used to study

differentiation, stratification and junction formation. However,

under 2D cell culture conditions, these cells will not form a

stratum corneum. Therefore, using epidermis and keratinocytes,

several key questions concerning polarity can be investigated,

such as how a life-long self-renewing tissue regulates the balance

between asymmetric and symmetric divisions and how this is

coupled to cell fate; how multicellular structures, such as hair

follicles, align in the plane of the tissue; and how apico-

basolateral polarity is established over multiple layers. In the

following sections, we highlight and discuss recent research on

the epidermis that provides novel insight into these questions.

Apico-basolateral polarity in a stratifyingepitheliumSimple epithelial cells polarize to establish two different

membrane domains – the apical and basolateral membranes –

which have distinct structural and functional characteristics and

are separated by the apical junctional complex (Niessen and

Gottardi, 2008). This complex consists of tight junctions, which

form an ion and size barrier and provide a fence that prevents

mixing of apical and basolateral membrane components,

adherens junctions and desmosomes (Fig. 1A). Apico-

basolateral polarity is essential for the formation of a protective

barrier, and for vectorial functions, such as the directed secretion

of components or transport of fluids. The establishment of apico-

basolateral polarity in simple mammalian epithelial cells is

achieved by complex agonistic and antagonistic interactions of

cell polarity protein complexes that allow for the spatial targeting

of polarity complexes to different domains within the cell

(Fig. 1A) (Nelson, 2009; Roh and Margolis, 2003; St Johnston

and Ahringer, 2010).

The stratifying epidermis is not a classically polarized

epithelium in which tight junctions separate basolateral and

apical membrane proteins and lipids. Instead, the epidermis

establishes polarity along the basal to apical axis of the tissue,

with the stratum granulosum forming the viable apical boundary

(Fig. 1B). The formation of the uppermost layer, the stratum

corneum, depends on the fusion of lamellar bodies (LBs) and

keratohyalin granules with the more apical plasma membranes at

the transition between stratum granulosum and corneum layers.

Interestingly, functional tight junctions are found in the stratum

granulosum and, as in simple epithelia, these might serve as a

fence that is necessary for restricting ‘apical’ targeting of protein

and lipid vesicles at the transition between the granular and

cornified layer.

The mechanisms that regulate the formation of stratifying

apico-basolateral tissue polarity are largely unknown. If

mechanisms similar to those in simple epithelia are in place,

the mutual antagonistic actions of polarity complexes have to be

established over several cell layers. A relatively simple system

could consist of counter-gradients of mutually inhibiting

complexes over the basal–apical axis of the epidermis

(Fig. 1B). From C. elegans to humans, in simple epithelia, the

formation and maintenance of intercellular junctions and apical

membrane domain identity is tightly linked to the activity of cell

polarity proteins, such as Par4 (liver kinase B1 in mammals,

LKB1), the Par–aPKC complex and the Crumbs homolog 1

(Crb1) complex (Goldstein and Macara, 2007; Nelson, 2003;

Bulgakova and Knust, 2009). Loss of either Crb1 or LKB1 does

not obviously impair epidermal barrier function, suggesting that

these proteins are dispensable, although their barrier properties

Table 2. Planar cell polarity proteins

Original symbol Mammalian symbol Function

Fz (Frizzled)a FZ3 7-TM receptorFZ6

Dsh (Dishevelled)a DVL1, DVL2, DVL3PDZ, DIX and DEP domain scaffold

Fmi (Flamingo), Starry knighta CELSR1 7-TM proteinVang (Van Gogh), Strabismusa VANGL1 4-TM protein

VANGL2 (looptail mutant)Dg (Diego)a Diversin (ANKRD6) Ankyrin-repeat containing scaffold

Inversin (INVS)Ft (Fat)a FAT4 Cadherin or TM proteinDs (Dachsous)a ND Cadherin or TM proteinFj (Four-jointed) ND TM serine/threonine kinasePk (Prickle)a PRICKLE1, PRICKLE2 LI- and PET-domain-containing protein

aIdentified in Drosophila.ND, not determined; TM, transmembrane protein.

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were not specifically assessed in these studies (Gurumurthy et al.,

2008; van de Pavert et al., 2004). Interestingly, the small GTPase

Rac and aPKC activity are necessary for tight junction barrier

function in stratifying keratinocytes in vitro (Helfrich et al., 2007;

Mertens et al., 2005). In addition, loss of cell adhesion molecules,

such as E-cadherin (Tunggal et al., 2005) or CD44 (Kirschner

et al., 2011), impairs skin barrier function that is associated with

an altered activity or localization of the Par–aPKC complex and,

in the case of CD44, with the loss of apical LBs. Taken together,

these results suggest that adhesive contacts and polarity signal

pathways cooperate in the skin in a similar manner to those in

simple epithelia to drive epithelial barrier function.

Regulation of cell fate and stem cell behavior inthe epidermisCell division not only generates daughter cells, but through the

control of orientation can also regulate their position within the

tissue, and/or their cell fate. Cells can divide either symmetrically

(symmetric cell division, SCD) resulting in two daughter cells

with similar fate, or asymmetrically (asymmetric cell division,

ACD) leading to two daughter cells with differential fate

(Fig. 3A,B). Organisms use this process not only to generate

different cell types during development but also for tissue

homeostasis and regeneration to replace cells that are turned over

or lost (Knoblich, 2010). For example, different populations of

stem and/or progenitor cells use ACD to allow for self-renewal

and differentiation at the same time (Farkas and Huttner, 2008).

One of the mechanistically best understood types of ACD

occurs in the Drosophila neuroblast, in which all divisions are

asymmetric, generating another neuroblast progenitor and a

ganglion mother cell. Neuroblast ACD requires the differential

partitioning of fate-determining proteins (Fig. 3A) (Knoblich,

2008), which is achieved by differential polarization of two

opposite membrane domains within the cell coupled with correct

positioning and orientation of the mitotic spindle. This will

ensure the appropriate partitioning of fate-determining factors

Fig. 3. Mechanisms of asymmetric cell division. Schematic overview of asymmetric localization of polarity proteins and spindle orientation regulators during

asymmetric cell division in Drosophila and in the interfollicular epidermis, illustrating that similar molecular mediators are involved in the establishment of

asymmetric cell divisions of neuroblasts and keratinocytes. (A) Asymmetric cell division (ACD) in Drosophila neuroblasts. The apical aPKC–Baz–Par6 complex

is connected to the Pins–Ga1–MUD complex through Inscuteable (Insc). This complex directs the asymmetric basal localization of the cell fate

determinants Numb, Brat and Prospero. GMC, ganglion mother cell. (B) ACD in the developing IFE. ACD contribute to stratification by producing one basal,

proliferating cell (light green) and one suprabasal cell (dark green), whereas symmetric cell divisions (SCD) result in two daughter cells residing in the basal layer.

aPKC–Par3, INSC and Ga1–LGN–NUMA–DCTN1 localize to one side of the dividing cell and are important for the establishment of epidermal ACD, as reported

for their Drosophila homologs in neuroblast ACD. Suprabasal activity of the Notch signaling pathway (indicated by nuclei positive for HES1, a well-known Notch

target) are crucial for the regulation of this process. (C) Effects of deletion or overexpression of molecular mediators on the ratio between ACD and

SCD and protein localization in the developing IFE. Epidermal deletion of aPKCl (left panel) results in an increase in perpendicular spindle orientation in the

epidermis. Forced induction of mouse INSC (middle panel) induces increased asymmetric spindle orientation in the epidermis shortly after induction (early),

whereas 3 days after induction, this effect is reversed (late) (Poulson and Lechler, 2010). Upon in vivo knockdown of LGN or NUMA (right panel), epidermal

basal cells are biased towards symmetric spindle orientation (Williams et al., 2011). Baz, Bazooka, Drosophila homolog of Par3; Par, partitioning defective;

aPKC, atypical protein kinase C; Pins, partner of Inscuteable (Drosophila homolog of LGN); Mud, mushroom body defect (Drosophila homolog of NUMA); Brat,

brain tumor; INSC, Inscuteable; NUMA, nuclear mitotic apparatus protein1; DCTN1, dynactin1.

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during cell division (Pearson and Bloom, 2004). Recently, many

authors have also demonstrated the importance of spindleorientation in regulating morphogenetic processes in mammals.

For example, ACD promotes the exit of one daughter cell from

the epicardium into the myocardium during heart morphogenesis(Wu et al., 2010), and regulate the differentiation of mammalian

neuronal progenitors (Zhong and Chia, 2008).

Asymmetric and symmetric divisions in the epidermis

In stratifying epithelia, spindle orientations that are either in

parallel or perpendicular to the basement membrane were firstobserved in the IFE (Smart, 1970), and later in the cornea

(Lamprecht, 1990), hair follicles (Blanpain and Fuchs, 2009;

Zhang et al., 2009) and in sebaceous gland development(Frances and Niemann, 2012) (Fig. 3B, Fig. 4). In the cornea,

perpendicular cell divisions are associated with a differential size

and morphology of the daughter cells (Lamprecht, 1990),suggesting that these divisions are asymmetric ones that

promote differential cell fate, whereas parallel divisions are

symmetric and produce two basal daughters that would increasethe surface area of the cornea. Lechler and Fuchs subsequently

showed that the switch from predominant symmetric (parallel)

divisions to asymmetric (perpendicular) divisions coincides withthe onset of stratification, providing indirect evidence that ACD

promotes epidermal differentiation (Lechler and Fuchs, 2005).

Indeed, whereas the basal daughter remains positive for the basalcell marker keratin 14, the suprabasal daughter originating from

ACD expresses keratin 10, a marker for suprabasal cell identity

(Poulson and Lechler, 2010).

An elegant in vivo lentiviral knockdown system used to

interfere with the molecular machinery that determines spindlepositioning in Drosophila neuroblasts, such as microtubule-

associated dynein binding protein (MUD) and partner of

inscuteable (PINS), provided first mechanistic insight into how

ACD is established in the murine epidermis. For example,knockdown of the mammalian PINS homolog LGN (ten leucine-

glycine-asparagine tripeptides in its N-terminal region) or the

MUD homolog NUMA (nuclear mitotic apparatus protein),results in a strong decrease of ACD, concomitant with an increase

in SCD (Fig. 3C) (Williams et al., 2011). These changes are

accompanied by a thinner epidermis that has a decreased numberof suprabasal layers with impaired expression of differentiation

markers, as well as a slight increase in basal cell number. This

observation links key regulators of spindle positioning todifferentiation in mammalian epithelial cells.

Unlike in Drosophila neuroblasts, not all epidermal divisionsare asymmetric. Moreover, the decision to divide either

symmetrically or asymmetrically is not predetermined in single

epidermal progenitors (Poulson and Lechler, 2010), indicatingthat the microenvironment plays an important role in the overall

outcome of cell divisions. In agreement with this model, loss of

cell adhesive cues, such as b1 integrins or the adherens junctionmolecule a-catenin in mouse epidermis, results in a random

spindle orientation that leads to altered differentiation patterns

(Lechler and Fuchs, 2005).

Polarity proteins in asymmetric and symmetric celldivision in skin

What is the importance of polarity signaling in regulating

asymmetric divisions in mammals? In Drosophila neuroblasts,

the initial polarization cue comes from the apical enrichment ofthe polarity proteins Par3, Par6 and aPKC. This apical

distribution is essential for asymmetric localization of cell fate

determinants, which is coupled to spindle orientation by bindingto the adaptor protein Inscuteable (Insc) (Fig. 3A). Insc then

recruits a protein complex consisting of the heterotrimeric G

Fig. 4. Symmetric and asymmetric divisions in different

compartments of the epidermis. In addition to ACD

occurring in the IFE (see Fig. 2B), ACD-like divisions have

been observed in the region where hair follicle stem cells

move outwards of the bulge region (hair germ) suggesting

that they fuel hair follicle anagen growth (black arrows),

whilst replenishing bulge cells. By contrast, inside the bulge,

symmetric cell divisions serve to replenish the stem cell pool

during anagen phases of the hair cycle. Asymmetric

divisions are also found in the bulb of the anagen hair

follicle positioned close to the dermal papilla and might

drive differentiation of the different hair follicle layers. BM,

basement membrane; SG, sebaceous gland; DP, dermal

papilla.

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protein a1-subunit (Ga1), PINS and MUD, which providesattachment sites for astral microtubules (Knoblich, 2010).

Polarized distribution of the aPKC–Par complex is inheritedfrom the epithelial cell, from which the neuroblast arose afterdelamination (Prehoda, 2009). Similarly, in both mouse neuronsand in the epidermis, PAR3 and aPKC show an apical

distribution that is independent of cell division (Lechler andFuchs, 2005). In the epidermis, this apical polarity might havebeen inherited from the polarized single layer epithelium before

the onset of stratification. Whereas in Drosophila neuroblasts theapical membrane domain determines the future neuroblast stemcell, in the epidermis, the aPKC- and Par3-enriched domain

marks the future differentiated suprabasal daughter cell (Poulsonand Lechler, 2010). In addition, Par3 and aPKC also show anapical localization in cells that undergo symmetric divisions,indicating that differential Par3 and aPKC localization alone is

not sufficient to drive ACD. In line with this observation is thefinding that the mammalian PAR–PAR6–aPKC complexregulates spindle positioning in SCD, as recently demonstrated

in 3D cell culture using knockdown approaches (Durgan et al.,2011; Hao et al., 2010).

In the developing mammalian brain, either loss or

overexpression of PAR3 increases SCD, but whereas PAR3overexpression increases the number of cells with progenitorradial glial cell fate, knockdown of PAR3 results in a greater

number of cells with differentiated fate (Bultje et al., 2009).Interestingly, this indicates that levels of PAR3 might directlydetermine cell fate. Similarly, loss of LGL1 in mice causes braindysplasia as a result of an increase in SCD (Klezovitch et al.,

2004). Thus, as in lower organisms, LGL1 and PAR3 in the brainare essential for ACD and work in an antagonistic manner toregulate differential cell fate. It is not yet clear whether PAR3

and LGL1 play similar roles in the developing epidermis.

The role of the two mammalian isoforms of aPKC – aPKCland aPKCf – in the regulation of ACD and SCD is more

controversial. Knockdown of aPKCl in ex vivo cultured mouseembryos alters cell fate (Dard et al., 2009), whereas in vivoinactivation of either aPKCl in developing neurons (Imai et al.,2006) or both mammalian aPKC isoforms in the hematopoetic

system (Sengupta et al., 2011) does not alter cell fate, suggestingthat, in mammals, aPKCs do not control ACD and thereby cellfate in vivo. By contrast, epidermal inactivation of aPKCl results

in an increase in asymmetric divisions in the interfollicularepidermis and the hair follicle that is associated with altereddifferentiation in both epidermal compartment (M.T.N., J. Scott

and C.M.N., unpublished results). This implies that aPKCl mightin fact be a negative regulator of asymmetric spindle orientation(Fig. 3C).

Knockdown of NUMA or LGN in the epidermis does not

affect aPKC or PAR3 localization (Williams et al., 2011)(Fig. 3C). A similar observation was made in mice with anepidermal deletion of serum response factor (SRF), a

transcription factor that activates gene expression of a range ofactin regulators (Luxenburg et al., 2011). In these mice, spindlepositioning is random and is accompanied by random cortical

localization of LGN, even though PAR3 remains apical(Luxenburg et al., 2011). Together, these data suggest that,similar to the process in neuroblasts, aPKC and PAR3 either act

upstream of LGN and NUMA, or that the apical localization ofthese two complexes serves different functions; for example, thePar–aPKC-mediated localization of thus far unknown cell fate

determinants versus LGN–NUMA-mediated spindle orientation.

SRF loss also alters the basal cell architecture that is associatedwith reduced cortical localization of the actinomyosincytoskeleton and the phosphorylated forms of the actin-

associated proteins ezrin, moesin and radixin (collectivelytermed ERM). Interestingly, loss of the PAR3 binding proteinmerlin (also known as NF2), an ERM-related protein, also resultsin altered spindle positioning that is accompanied by impaired

differentiation (Gladden et al., 2010). Thus, perhaps PAR3regulates the recruitment of spindle position regulators to theapical membrane through regulation of ERM–merlin and

actinomyosin.

In Drosophila neuroblasts, the Par–aPKC complex is notabsolutely required for correct spindle orientation, because flieshave an alternative mechanism that involves microtubules and

the MUD complex, which is activated in telophase when theapical determinant complex is either missing or does not functionproperly (reviewed by Knoblich, 2010). Recent data indicate that

the epidermis might also correct potential imbalances in divisionorientation, albeit through a very different mechanism. Epidermaloverexpression of mouse INSC initially promotes asymmetric

divisions, but this effect is reversed upon prolonged INSCexpression, and is accompanied by the dissociation of NUMAfrom the apically localized INSC and LGN proteins (Fig. 3C)

(Poulson and Lechler, 2010). This result suggests that epidermalprogenitors can sense and correct disturbances in the balance ofACD versus SCD (Ray and Lechler, 2011). Taken together, theserecent findings suggest that although the core machinery that

controls ACD in Drosophila is conserved in the mammalianepidermis, there are also important differences with regard to theregulation of ACD and SCD.

Asymmetric divisions and cell fate determination:outstanding questions

One important question that remains to be answered is whether

epidermal ACD is directly coupled to the asymmetric segregationof cell fate determinants, as is the case in Drosophila neuroblasts,and/or whether differential cell fate is achieved through unequal

positioning of cells, which then receive diverse signals from thelocal environment to regulate their fate. Some evidence for thelatter possibility is provided by the observation that the strongdecrease in ACD upon in vivo knockdown of LGN in the

epidermis is associated with a reduction in Notch signaling,which is necessary in suprabasal layers to promote epidermaldifferentiation (Williams et al., 2011).

Another important remaining question is whether asymmetricdivisions regulate cell fate decisions of stem or progenitor cellbehavior in adult skin. Lineage-tracing studies in mice combinedwith mathematical modeling imply that random SCD and ACD

occur in the adult IFE (Clayton et al., 2007). However, it has beendebated whether these cells truly represent long-lived stem cells.In mice, the hair follicle bulge harbours the most pluripotent stem

cells and lineage tracing experiments in this system suggest thatsymmetric divisions replenish these cells (Petersson et al., 2011;Zhang et al., 2010). This is consistent with findings that hair

follicle stem cells do not oblige with the ‘immortal strand’hypothesis (Sotiropoulou et al., 2008; Waghmare et al., 2008).This proposes that stem cells asymmetrically segregate their

chromosomes to retain the same strand of DNA over severaldivisions in order to avoid accumulating DNA mutations.Progenitors located just below the bulge in the hair germ

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(Greco et al., 2009) can divide asymmetrically (Zhang et al.,2010) and some recent evidence suggest that these cells replenish

bulge stem cells while at the same time providing enough cells todrive anagen hair follicle growth (Hsu et al., 2011) (Fig. 4).Perpendicular spindles have also been observed in the hair bulb(Blanpain and Fuchs, 2009), suggesting that such asymmetric

divisions might regulate the differentiation of the various hairfollicle layers. One important caveat in all these studies is thattracing cell divisions cannot yet be correlated with tracing of the

differential cell fate. Nevertheless, indirect evidence from globalRNA expression data suggests that dividing stem cells in thebulge retain their stem cell signature in agreement with SCD for

these cells (Zhang et al., 2009). It will be important to furtherexamine the in vivo role of polarity proteins in the regulation ofcell fate in the epidermis and its appendages.

Orientation of hair folliclesThe orientation and alignment of hair follicles along the bodyaxis is an example of tissue polarity in the adult skin. In mice,

hair follicles and hairs are inserted with an angle of around 45–55˚ relative to the anterior–posterior (A-P) body axis (Fig. 2D).Unlike single-cell appendages, such as the hair on a Drosophila

wing or the stereocilia in the inner ear sensory hair cell, hairfollicles consist of hundreds of cells. The individual cell positionnot only needs to be aligned with their direct neighbours, but thecells of an individual hair follicle also have to act as a unit to

communicate their position with adjacent hair follicles to achievethis global anterior-posterior hair follicle pattern. Hair folliclemorphogenesis starts at embryonic day 14.5 (E14.5) with the

budding of a placode from the basal epidermal layer in responseto inductive signals from the dermis (Schneider et al., 2009).Initially, placode formation is symmetric with first signs of A-P

asymmetry observed later at the hair germ stage, whendeveloping follicles adopt their polarized position owing tobasal constriction of HF cells at the anterior side, concomitant

with posterior cells adopting a columnar shape (Fig. 2B). This isaccompanied by differential localization of several proteins eitherat the anterior or posterior side of the hair follicle (Devenport andFuchs, 2008).

Several recent papers have provided substantial evidence thatthis coordinated polarization of hair follicles is driven by theactivity of core PCP proteins. Upon loss of one of the PCP

transmembrane component, either mammalian FZ6 (Guo et al.,2004) or the mammalian Flamingo ortholog CELSR1 cadherin(Ravni et al., 2009), hair patterning is disturbed, resulting in hair

swirls (Fig. 2C). Closer analysis of FZ6-deficient mice indicatesthat hair follicle and shaft orientation is regulated by at least twosystems, an early-acting global orientation that depends on FZ6signaling and a later-acting FZ6-independent local PCP system

(Wang and Nathans, 2007). How is asymmetry established in theearly hair follicle? Recent data indicate that FZ6 probably actstogether with CELSR1 and VANGL2 in a non-cell-autonomous

fashion early in hair follicle morphogenesis to globally orient hairfollicles. One day before placode formation (E13.5), FZ6,VANGL2 and CELSR1 exhibit a distinct localization pattern,

with VANGL2 enriched on anterior basal membranes, FZ6 onposterior ones and CELSR1 on both (Devenport and Fuchs,2008), which is a similar pattern to that observed for these

molecules in, for example, Drosophila wing cells (Fig. 2A,B).Mutations in genes encoding CELSR1 or VANGL2 not onlydisturb anterior and/or posterior localization of VANGL2, FZ6

and CELSR1 (Devenport and Fuchs, 2008), indicating that theirlocalization is mutually dependent, but also prevent constriction

and hair follicle asymmetry (Fig. 2D). PCP proteins areinternalized during mitosis in a manner that is dependent onCELSR1, and segregate symmetrically upon division, after whichthey regain their polarized distribution (Devenport et al., 2011).

This mechanism probably serves as a protective mechanismagainst improper planar signaling across different cells and hairfollicles, when cells round up during mitosis. Prevention of their

internalization during development interferes with the orientationof hair follicles (Devenport et al., 2011). Together, these resultsindicate that the core FZ6–VANGL2 pathway regulates the

individual and global polarization of hair follicles.

Altered polarity – a hallmark of cancerHallmarks of cancer are unrestricted cell division, an altered

cyto-architecture and a gain of migratory and invasive capacities,all properties in which polarity proteins have a key role. Ingeneral, loss or reduced expression of polarity proteins (e.g.

LKB1, Scribble, DLG1, LGL2, DSH, PAR3 and Crumbs) hasbeen associated with tumor initiation and/or progression(Hawkins and Russell, 2008; Huang and Muthuswamy, 2010;

Humbert et al., 2008; Lee and Vasioukhin, 2008), suggestingthat they might have tumor suppressive function. The E6oncoprotein of human papillomavirus (HPV) strains associatedwith high carcinogenesis risk target human Scribble and DLG1

for degradation (Gardiol et al., 1999; Nakagawa and Huibregtse,2000), in agreement with such a tumor suppressive function. Inmice, loss of Scribble indeed results in prostate neoplasia

(Pearson et al., 2011). However, only LKB1 mutations havebeen directly linked to human cancer thus far (Jansen et al.,2009). Epidermal inactivation of LKB1 predisposes mice to non-

melanoma skin cancer, thus providing direct evidence that LKB1serves as a cell-autonomous tumor suppressor in the skin(Gurumurthy et al., 2008). Epidermal loss of PAR3 also

inhibits formation and growth of papillomas. However, suchmice are predisposed to the formation of keratoacanthomas (S.I.and J. G. Collard, unpublished). These tumors are thought toderive from a different cellular subpopulation within the

epidermis than papilloma (Perez-Losada and Balmain, 2003),thus suggesting that the cellular context might determine whetherPAR3 functions as a tumor promoter or suppressor.

An important question for the future is how altered polaritysignaling inhibits and/or promotes (skin) carcinogenesis. This islikely to occur on different levels. For instance, tumor initiation

might occur through alterations in the extent of ACD comparedwith SCD. In Drosophila neuroblasts, switching from ACD toSCD promotes unrestricted growth and allows for tumorformation (Januschke and Gonzalez, 2008). An increase in

ACD is therefore predicted to inhibit mammalian tumorigenesis,whereas an increase in SCD could promote carcinogenesis (Leeand Vasioukhin, 2008). Although this has not been rigorously

tested in mammals, it is interesting to note that overexpression ofPAR6 or aPKCi is associated with a range of human carcinomas(Fields et al., 2007; Nolan et al., 2008). In addition, expression

of a constitutive membrane-bound aPKC promotes symmetricdivisions and overgrowth in Drosophila neuroblasts (Lee et al.,2006). By contrast, loss of aPKCl almost completely prevents

tumorigenesis in either mouse models of colon cancer (Murrayet al., 2009) or lung carcinoma (Regala et al., 2009). PAR6–aPKC might also drive tumorigenesis through interactions with

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kinase receptors, because for example, transformation driven by

the tyrosine kinase ERBB2 depends on PAR6–aPKC (Aranda

et al., 2006). Similarly, many in vitro studies have shown that

polarity proteins have a crucial role in the assembly and

disassembly of cell–cell junctions (Feigin and Muthuswamy,

2009; Suzuki and Ohno, 2006), as well as in migration (Caddy

et al., 2010; Dow et al., 2007; Pegtel et al., 2007; Tscharntke

et al., 2007). This is not only essential for wound healing (Caddy

et al., 2010), but also is a key determinant for invasion of tumor

cells.

Conclusion and PerspectivesThe last decade has brought tremendous progress in our insights

into how cell and tissue polarity proteins regulate a range of

biological essential processes. Only recently, in vivo tissue-

specific mammalian models have become available that now

allow us to tease out the cell-autonomous and non-cell-

autonomous functions of these proteins in different tissues. As

highlighted here for the epidermis, these studies started to reveal

important insights into their function during morphogenesis.

Because many core polarity proteins have multiple mammalian

orthologs, a future challenge will be to identify their unique and

overlapping roles, as well as to determine tissue-specific

requirements and link them to human disease. In this context, it

is important to note that polarity protein functions not only

determine cell and tissue architecture, but have also been

implicated in the regulation of a variety of cellular pathways

that are crucial for growth, differentiation, metabolic activity and

innate immunity. Thus, polarity signal pathways could turn out to

be central integrators of cyto-architecture and tissue homeostasis.

AcknowledgementsWe would like to apologize to all colleagues whose original work wecould not cite because of space limitations. We would like to thankJeremy Nathans (Johns Hopkins University, Baltimore) for providingus with the images for Fig. 2C. We thank Susanne Vorhagen andRehan Villani for helpful discussions.

FundingC.M.N. is funded by the Deutsche Krebshilfe and the DeutscheForschunsgemeinschaft (DFG) [grant numbers SFB829 A1, Z2 andSFB832 A3 and Z3]. S.I. is supported by the DFG [grant numbersSFB829 and SFB832], and by the Stiftung Kolner Krebsforschung.

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