Deletion of the epidermis derived laminin γ1 chain leads to defects in the regulation of late hair morphogenesis

Anja Fleger-Weckmann1*, Yasemin Üstün1*, Jennifer Kloepper2, Ralf Paus3, Wilhelm

Bloch4, Zu-Lin Chen5, Jeannine Wegner6,7, Lydia Sorokin6,7, Lutz Langbein8, Beate

Eckes1, Paola Zigrino1, Thomas Krieg1,9,10 and Roswitha Nischt1

1Dermatology, University of Cologne, Germany 2Dermatology, University of Lübeck, Germany 3Dermatology, University of Manchester, UK

4German Sport University Cologne, Germany5Neurobiology and Genetics, Rockefeller University, New York, USA6Physiological Chemistry and Pathobiochemistry, University of Muenster, Germany7Cells-in-Motion Cluster of Excellence, University of Muenster, Germany8German Cancer Research Center, Heidelberg, Germany9Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany10Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Germany

* These authors contributed equally

Running title Epidermal laminin γ1 regulates late hair morphogenesis

Key words Skin, basement membrane, laminin-511, laminin-211, hair follicle

Address for correspondence:

Professor Thomas Krieg, MDDermatology, University of CologneKerpener Str. 62 phone +49-221-478 8224550931 Cologne, Germany Email thomas.krieg@uni-koeln.de

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AbstractLaminins are the most abundant non-collagenous basement membrane (BM)

components, composed of an α, β and γ chain. The laminin γ1 chain, encoded by

LAMC1, is the most abundant γ chain. The main laminin isoforms in the dermo-

epidermal junction (DEJ) are laminin-332, laminin-511 and laminin-211, the latter

being restricted to the lower part of hair follicles (HFs). Complete deletion of LAMC1

results in lethality around embryonic day 5.5. To study the function of laminin γ1

containing isoforms in skin development and maturation after birth, we generated

mice lacking LAMC1 expression in basal keratinocytes (LAMC1EKO) using the keratin

14 (K14) Cre/loxP system. This deletion resulted in loss of keratinocyte derived

laminin-511 and in deposition of fibroblast derived laminin-211 throughout the whole

DEJ. The DEJ in areas between hemidesmosomes was thickened, whereas

hemidesmosome morphology was normal. Most strikingly, LAMC1EKO mice showed

delayed HF morphogenesis accompanied by reduced proliferation of hair matrix cells

and impaired differentiation of hair shafts (HS). However, this deletion did not

interfere with early HF development, since placode numbers and embryonic hair

germ formation were not affected. Microarray analysis of skin revealed down

regulation of mainly different hair keratins. This is due to reduced expression of

transcription factors such as HoxC13, FoxN1, FoxQ1 and Msx2, known to regulate

expression of hair keratins. While the role of laminin-511 in signaling during early hair

germ formation and elongation phase has been described, we here demonstrate that

epidermal laminin-511 is also a key regulator for later hair development and HS

differentiation.

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Abbreviations: basement membrane (BM), bone morphogenetic protein (BMP) dermo-epidermal junction (DEJ), embryonic day (E), Forkhead box N1 (FOXN1), hair follicle (HF), hair shaft (HS), Homeobox C13 (HOXC13), immunofluorescence (IF), inner root sheath (IRS), keratin 5 (K5), keratin 14 (K14), keratin associated proteins (KAPs), Lymphoid enhancing factor (Lef1), Homeobox msh-like 2 (MSX2), polymerase chain reaction (PCR), postnatal day (P), reverse transcriptase (RT), Sonic hedgehog (Shh), transient amplifying (TA), transcription factor (TF)

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Introduction

BMs are highly structured extracellular matrices that form a dynamic interface

separating and connecting simultaneously epidermal with mesenchymal

compartments. Its components are regulators of cellular processes essential for skin

integrity as illustrated by several acquired and inherited diseases [1], [2]. The major

components of the DEJ are members of the laminin, nidogen, collagen IV and the

heparan sulfate proteoglycan (perlecan and agrin) families [3], [4].

Laminins are the most abundant non-collagenous structural components of BMs and

play important roles in tissue morphogenesis and homeostasis by regulating cell

survival, differentiation, migration and adhesion [5], [6]. Furthermore, they link the BM

to epithelial transmembrane receptors, primarily β1 and β4 integrins [7]. Laminins are

heterotrimeric proteins composed of α, β, and γ subunits [8]. Currently, 5 α-, 3 β- and

3 γ-subunits have been identified in mice and humans forming the 18 known laminin

heterotrimers [5], [9]. The different isoforms exhibit tissue and developmental

specificity [10], [11], [12], reflecting their diverse functions in vivo.

Laminins in the skin have been implicated in the regulation of HF development,

driven by a series of epithelial-mesenchymal crosstalk [13]. HF development is

initiated by the induction of epithelial hair placodes by the first message of the

underlying mesenchyme [14] and proceeds with the down growth of hair germs into

the dermis to form HFs [15]. Induced by the epithelial message derived from

elongating HFs, dermal papilla cells condensate and become surrounded by the hair

bulb. Signals from dermal papilla cells stimulate proliferation of adjacent hair matrix

cells of the HF. During HF differentiation, matrix cells produce progenitors that

differentiate and give rise to HS and inner root sheath of the HFs [16], [17], [18], [13].

As the cells move distally into the different HF compartments, they start to

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differentiate, visible by the expression of characteristic marker proteins (keratins).

Differentiation of matrix cells is regulated by a number of factors, such as

Wnt/Lymphoid enhancing factor 1 (Lef1) [19], [20], fibroblast growth factor 7 (Fgf7)

[21] and members of the bone morphogenetic protein (BMP) family [22], [23]. These

are important in HF proliferation and differentiation during hair morphogenesis.

However, the role of extracellular matrix proteins, which provide critical anchorage to

the cells, in this process is not elucidated.

The laminin γ1 chain is the most ubiquitously expressed γ subunit, present in 10 of

the 18 known laminin isoforms [5]. Previous studies showed that laminin γ1 is a

prerequisite for the formation of an embryonic BM, since LAMC1 complete knock out

mice die at embryonic day (E) 5.5 [24]. Expression of laminin-111 and -511 is already

detected at the morula stage in mouse development [25]. At the DEJ, laminins are

synthesized by both dermal [26], [27] and epidermal cells [28]. The main secreted

laminin variants at the DEJ are laminin-332 and laminin-511. Depending on the

developmental stage, laminin-211 is also present in the lower part of the HFs [29],

[9]. During HF elongation, the laminin composition of the BM undergoes changes:

while laminin-511 expression pattern is not altered, laminin-332 [30] and laminin-111

[31] are downregulated. Previous studies have shown the importance of laminin-511

in early embryonic hair germ elongation. The loss of laminin-511 in transgenic mice

resulted in a reduced follicular proliferation, thus in a reduced number of hair germs

after regression of the HFs [25]. Further known laminin γ1 chain containing isoforms

in the skin are laminin-311, and -321, however, their presence in murine skin is

discussed controversially [9].

To study the function of laminin γ1 chain containing isoforms in skin development

and maturation in later stages, and to avoid early embryonic lethality, we generated

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mice that do not express LAMC1 in basal keratinocytes from early developmental

stages onwards (referred to as LAMC1EKO) using the K14 Cre/loxP system [32].

This report demonstrates that epidermal derived laminin γ1 chain is a key regulator

for late hair morphogenesis and differentiation. We showed that the deletion of

epidermal laminin γ1 resulted in the loss of keratinocyte derived laminin-511 and in

ectopic deposition of fibroblast derived laminin-211 in the whole DEJ. These changes

in laminin composition led to ultrastructural defects of the dermo-epidermal BM zone

between hemidesmosomes and in delayed hair growth, accompanied by reduced

proliferation of hair matrix cells and defects in hair differentiation. Furthermore, our

studies suggest an important role of the BMP-MSX2-HOXC13-FOXN1 signaling axis

in regulation of the processes required for HF differentiation and proliferation.

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Results

Depletion of keratinocyte specific laminin γ1 from the dermo-epidermal BM

The main laminins in the murine dermo-epidermal BM are laminin-332 and the

laminin γ1 chain containing isoform laminin-511 and, in the lower part of the HF,

laminin-211.

To analyze the functional impact of the γ1 chain containing laminins in late hair

development, homozygous floxed laminin γ1 mice [33] were crossed with a

transgenic mouse strain expressing Cre-recombinase under the control of the human

K14 promoter [32]. This results in the mouse strain LAMC1EKO which lacks laminin γ1

chain expression and secretion by basal keratinocytes. The human K14 promoter

becomes active at around E14.5, and activity in postnatal mice is restricted to the

mitotically active basal keratinocytes, the outer root sheath of the HFs and the oral

epithelium [34]. After birth, homozygous LAMC1EKO mice were easily distinguished

from their control littermates by syndactyly of the hind limbs (Fig. 1a), as well as by

growth retardation, reduced body size and delayed appearance of the hair coat. Of

151 mice analyzed, hind limb syndactyly was observed in 98.7% of mice carrying the

Cre-transgene and only 1.3% of mice displayed partial hind limb syndactyly. Delayed

hair growth was observed in 100% of LAMC1EKO mice. Macroscopically, the observed

hair phenotype did not reveal a high variability in severity, and it correlated with

syndactyly and reduced body growth.

To analyze the influence of epidermal laminin γ1 chain depletion on the development

of the skin at the molecular level, we performed immunofluorescence (IF) stainings

for different laminin chains on skin sections of LAMC1EKO and control mice. Laminin

composition of the DEJ was analyzed at early and late time points of hair

morphogenesis (E18.5, P1 and P10) before entry into first catagen phase at P16. At

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all time points investigated, skin sections of LAMC1EKO mice revealed reduced

deposition of the laminin γ1 chain in the DEJ, but not a complete loss, when

compared to control skin, indicating that laminin 1 in the DEJ was produced by other

cell types, such as fibroblasts. Staining with antibodies directed against different

laminin α chains revealed absence of laminin α5 chain in the DEJ of LAMC1EKO skin,

while staining of BMs of capillaries, arterioles and nerves was still detected. Laminin

α2 deposition was restricted to the lower part of the HFs in control skin, however,

mutant skin sections revealed prominent continuous laminin α2 staining of the

dermo-epidermal BM (Fig. 1b). Our findings confirm that laminin-511 at the DEJ is

synthesized by keratinocytes, while fibroblasts produce the laminin γ1 chain

containing laminin-211 as previously reported [27], [35]. This result is consistent with

data obtained by in situ hybridization (Fig. S1a) and with reverse transcriptase (RT)-

polymerase chain reaction (PCR) using RNA isolated from fibroblasts or

keratinocytes from control or LAMC1EKO skin (Fig. S1b). By contrast, the localization

of the laminin α3 and α4 chains and deposition of laminin-332 was not changed in

LAMC1EKO mice (data not shown). Since binding of the laminin γ1 chain to nidogen-1

and -2 [36] mediates the connection of laminins to the collagen IV network and to

other proteins like perlecan, we also investigated the distribution of these major BM

molecules. However, IF stainings did not reveal changes in the localization of these

proteins at the DEJ of LAMC1EKO skin (data not shown).

In agreement with our staining results, Western blot analysis of serum-free

supernatants of primary control and mutant keratinocytes showed loss of both

laminin γ1 and α5 chains in the supernatants of mutant keratinocytes, indicating that

laminin α5 chain is not secreted in the absence of laminin γ1 (Fig. 1c). This finding is

in concordance with the concept that laminin assembly into trimeric molecules takes

place intracellularly and that only laminin trimers are secreted [37]. By Western blot

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analysis using keratinocyte lysates, we could indeed confirm that laminin α5 chain,

although not secreted by keratinocytes of LAMC1EKO mice, was still present in these

cells, whereas no intracellular laminin γ1 was detected. This was different in

fibroblasts, which secreted the laminin γ1 chain normally, as expected from previous

studies [27], [35] (Fig. 1c).

Ultrastructural analysis of skin sections at P1 revealed that a discontinuous dermo-

epidermal BM in areas between hemidesmosomes of LAMC1EKO skin. Partially

preserved lamina densa between the hemidesmosomes appeared diffuse with less

compact extracellular matrix deposits when compared to control littermates.

However, hemidesmosome formation per se was not affected in the mutants.

Furthermore, skin of P9 mutant mice revealed alterations of the epidermal

ultrastructure with less compact packing of basal keratinocytes and enlarged

intercellular spaces. Basal keratinocytes showed apoptotic features such as pyknotic

nuclei reflecting chromatin condensation (Fig. 1d).

HF morphogenesis is impaired after depletion of laminin γ1 chain expression in

basal keratinocytes

Apart from syndactyly of the hind limbs, the most striking finding in LAMC1EKO mice

was the complete absence of a hair coat at P7 to P10 when control mice were fully

covered with pigmented hair. Mutant mice developed a sparser hair coat that covered

the mice by P18, and pigmentation of HSs was not observed before P15. To

elucidate the effect of the laminin γ1 chain containing laminins on hair development,

we characterized HF morphology and HF developmental stages in control and

LAMC1EKO skin sections at P1 and P10.

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Changes in HF morphology were assessed by hematoxylin and eosin stainings of

longitudinal back skin sections from mice at P1 and P10 (Fig. 2a). At P1, mutant skin

morphology did not reveal any major differences compared to control animals (Fig.

2a). However, at P10, HF orientation, length and structure were affected in the

mutant skin (Fig. 2a). While HFs of control mice were uniformly arranged at an angle

of 30° to the epidermal surface, the orientation of HFs in mutant skin was

disorganized, showing varying angles of HFs between 40° and 90°. Furthermore,

mutant mice lacked HSs with the typically striped pigmentation pattern and HFs that

were pigmented revealed a uniform light-brown color. HFs in mutant skin were much

shorter and did not grow deeply into the subcutis, rather they were curved and

thickened in some areas when compared to HFs in control skin (Fig. 2a). At the

ultrastructural level, the follicular BM of LAMC1EKO mice at P3 revealed thickened BM

areas compared to control mice (Fig. 2b).

Morphologically, HF development proceeds through 8 stages, divided into 3 phases:

the induction phase (stage 0 and 1), the organogenesis phase (stage 3-5) and the

cyto-differentiation phase (stage 6-8) [18], [13]. Deletion of laminin γ1 chain timely

correlates with placode formation (E13.5). The HF stages at P1 and P10 were

compared in control and mutant mice. At P1, in control animals 40% of the back skin

HFs were in stage 6. In contrast, only 8% of HFs in mutant mice were at this stage.

At P10, almost all HFs were in stage 8 in controls, whereas in mutant mice only half

of the HFs reached this stage (Fig. 2c), indicating a delay in hair growth of

approximately 8 days compared to control littermates.

To determine if the delay in HF morphogenesis results from reduced proliferation of

hair matrix cells that give rise to the growing HS, the number of proliferating hair

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matrix cells below Auber’s line, i.e. the widest diameter of the dermal papilla [38],

was determined by Ki67 staining (Fig. 2d). Indeed, numbers of proliferating matrix

cells were significantly reduced (p<0.005) in LAMC1EKO as compared to control HFs

(Fig. 2e).

In the mouse, HF morphogenesis starts at E13.5 with formation of the placode and

proceeds with its progression to hair germ to hair peg. Embryos at E16.5 exhibit all

three HF precursors, which were assessed in the skin of LAMC1EKO and control

embryos by performing IF co-staining for P-cadherin and K14 (Fig. 3a, b). The total

number of HF precursors at the distinct stages was determined and revealed no

difference between control and LAMC1EKO mice (Fig. 3a). Quantification of embryonic

hair placode numbers at day 16.5 (Fig. 3c) and mature HF numbers in 8-week old

adult mice (Fig. 3d) revealed comparable numbers in control and LAMC1EKO skin.

Together, these data indicate that K14-Cre mediated epidermis specific deletion of

the laminin γ1 chain does not significantly interfere with HF induction.

Differential gene expression after keratinocyte specific laminin γ1 depletion

To identify genes whose expression were affected by depletion of LAMC1 expression

in keratinocytes, the mRNA expression profile of the epidermis and dermis from

LAMC1EKO split skin was analyzed at E18.5 and P6. At P6, HFs have already invaded

deeply into the dermis and subcutis, thus, the dermal compartment of split skin can

contain parts of HFs resulting from incomplete separation of the two compartments.

Analyzing split skin from 2 male control and mutant mice at P6 revealed differential

expression of 596 genes in the dermis and 373 genes in the epidermis (p<0.05;

>±1.5-fold) of LAMC1EKO mice compared to age-matched control samples. Among the

genes with altered expression in the dermis, 209 genes were specifically

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downregulated and 348 were upregulated. In the epidermis of LAMC1EKO mice,

expression of 77 genes was specifically upregulated, while expression of 257 genes

was specifically downregulated (Fig. S2). Most of the upregulated genes were

involved in immunological processes and were mainly detected in the dermis. The

most strongly downregulated genes in the LAMC1EKO skin included genes encoding

hair keratins or keratin associated proteins (KAPs) (listed in Fig. 4a). The analysis

further revealed downregulation of transcription factors known to directly or indirectly

regulate expression of hair keratins found in the cortex, medulla and cuticle layers of

the HS (listed in Fig. 5a). Altered expression of these genes was confirmed by

analysis of the respective proteins (see below).

The expression profile of split skin (into dermis and epidermis) at E18.5 failed to

reveal major differences between LAMC1EKO and control skin. In total, ten gender-

unspecific genes exhibited significant changes in expression. Among these, we

identified a group of upregulated immune-associated genes (data not shown).

Differentiation of the HS is impaired in LAMC1EKO HFs

Rapidly dividing keratinocytes in the hair matrix form seven concentrically arranged

layers of HFs [39]. Since we had identified reduced proliferation of hair matrix cells in

the LAMC1EKO mutants (Fig. 2d, e) we asked whether this defect might interfere with

HF differentiation. We therefore performed IF stainings of skin sections using hair

keratin antibodies specific for different HS compartments as well as for HF-specific

epithelial keratins [40], [41]. Immunostainings of P6 skin sections did not reveal

differentiation defects of the companion layer (K75) and the IRS (K28) of LAMC1EKO

mice. By contrast, the development of the HS, which itself comprises medulla (K81),

matrix and/or cortex (K33, K35, K81, K85, K86) and cuticle (K35, K82, K85), was

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affected, as shown by the altered expression patterns of the HS keratins in the

mutant mice (Fig. 4b). This result agrees with our array analysis, which revealed

downregulation of these keratins at the transcriptional level (Fig. 4a).

In addition to keratins, microarray analysis of LAMC1EKO split skin has also revealed

downregulation of the transcription factors Homeobox C13 (HOXC13), Forkhead box

N1 (FOXN1), Forkhead box Q1 (FOXQ1) and Homeobox msh-like 2 (MSX2), all

known to directly or indirectly regulate expression of hair keratins found in the cortex,

medulla and cuticle layers of the HS [42], [43], [44] (Fig. 5a). Downregulation of the

transcription factors Msx2, FoxN1 and HoxC13 was confirmed at the protein level by

IF and/or by immunoblot analysis. IF analysis showed reduced FoxN1 protein in the

HS and IRS of mutant HFs at P6 and P10. Further, staining for HoxC13 revealed a

reduced amount, as well as an impaired localization of the protein in the companion

layer and matrix region of HFs of LAMC1EKO animals (Fig. 5b). Reduced FoxN1,

HoxC13 and Msx2 expression was further confirmed by immunoblot analysis using

skin lysates of mutant and control mice (P6 and P10) (Fig. 5c). In addition to these

transcription factors, the regulatory proteins Lef1 and BMP2, known to be important

for HF proliferation and differentiation [19], [20], [22], [23] were also downregulated

(Fig. 5a). As a consequence of reduced BMP signaling activity in LAMC1EKO skin, we

detected reduced pSmad1/5 expression at P6 and at P10 (Fig. 5d). A working model

for the effect of the identified regulatory molecules on HF morphogenesis is shown in

Fig. 6.

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Discussion

This study is the first to describe the role of the epidermal laminin γ1 chain, the most

abundant laminin γ chain, as an important regulator for late hair morphogenesis and

differentiation. As previously reported, the complete deletion of laminin γ1 results in

embryonic lethality of mice preventing skin and hair analysis after birth [24]. Here, we

generated mice with LAMC1 deficiency in basal and outer root sheath keratinocytes

(LAMC1EKO) induced during late embryogenic development, using the K14 Cre/loxP

system.

The main laminin γ1 containing isoforms in the DEJ are laminin-511 [9] and laminin-

211, the latter found in the lower part of the HFs. Our analysis showed that

LAMC1EKO mice lack laminin α5 chain deposition at the DEJ, confirming that laminin-

511 is deposited by keratinocytes [35] and moreover that laminin molecules are

secreted only following heterotrimerization, while assembly takes place intracellularly.

Furthermore, laminin α2, normally restricted to the lower part of the HFs was

ectopically deposited throughout the whole BM of LAMC1EKO skin, confirming

fibroblasts as the source of laminin-211 expression and secretion [27] (see also Fig.

1c). Consistent with these findings, laminin α2 has already been identified as a

product of mesenchymal origin in previous studies [27], [9].

Since the laminin γ1 chain was proposed to play an essential role in the integration of

other major BM molecules such as collagen IV, perlecan and nidogens, their

distribution could have been affected by the inactivation of LAMC1. However, these

molecules were normally distributed in LAMC1EKO skin, implicating that fibroblast

derived laminin γ1 chain is sufficient for their integration into the DEJ.

The phenotypic changes in LAMC1EKO mice, however, demonstrate that the

ectopically deposited, fibroblast derived laminin-211 is not fully able to functionally

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compensate for the lack of laminin-511. The described phenotypes can be caused

either by loss of laminin-511 or by ectopic deposition of laminin-211. In the follicular

BM, particularly in the area surrounding the DP, laminin-211 is present during HF

morphogenesis in both, control and mutant skin. Thus, its expression should not alter

the HF phenotype of LAMC1EKO mice, but the loss of laminin-511 is causal for the

observed defects. However, there is an altered distribution of laminin-211 in mutant

HFs, so it may be the local balance of these laminins that is necessary for proper HF

development. By contrast, ultrastructural defects of interfollicular epidermis and the

interfollicular DEJ can be attributed to the combined loss of laminin-511 and the

ectopic presence of laminin-211, the latter being absent from control interfolliclular

epidermis.

Laminins mediate outside-in signaling (intracellularly) through transmembrane

receptors such as integrins [45]. Laminin-211 and -511 are recognized by partially

overlapping sets of cell surface receptors including integrins α6β1 and α7β1 [46],

[47], [5]. Dystroglycan is also expressed by epidermal cells [48] and binds with high

affinity to laminin-211 [49], but weakly, if at all, to laminin-511 [50]. An abundant

receptor for laminin-511 in the skin is integrin α3β1, which, however, does not bind to

laminin-211 [51], [52]. Therefore, the ultrastructure of the lamina densa was markedly

abnormal in LAMC1EKO mice, possibly due to the ectopic deposition of laminin-211,

which is not able to bind to integrin α3β1. This results in rounding-off of basal

keratinocytes, likely due to a partial loss of cell polarity. However, hemidesmosomal

adhesion of basal keratinocytes to the underlying BM was not disturbed,

demonstrating that the interaction of laminin-332 with integrin α6β4 is sufficient to

prevent epidermal detachment and blistering.

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Of note, LAMC1EKO mice exhibited delayed HF morphogenesis and a defect in HF

differentiation. Findings of previous studies on mice with ablation of laminin α5 [25],

integrin β1 [53], [54] and integrin α3 [55] support our hypothesis that late hair

morphogenesis is affected by the lack of interaction between laminin-511 and integrin

α3β1, which has a direct role in proliferation of hair matrix cells. For laminin α5

deficient mice it was shown that embryos have reduced numbers of hair germs [25].

However, we found that hair placode formation was not affected in our mutant mice

due to the later onset of the K14 promoter and induction of LAMC1 deletion at around

E14.5 (data not shown) [34]. Mice with an epidermis specific deletion of laminin α5

induced by keratin 5 (K5)-Cre show a delay of two days in the appearance of hair

after birth. In these mice, HF morphology was not disturbed, but HF density was

slightly reduced (L. Sorokin, personal communication). This differed from our results

and might be explained by different time of onset of K5 and K14 promoters.

Interestingly, both studies found that loss of laminin-511 at the DEJ was associated

with an upregulation of laminin α2 in this region. In addition, laminin α4 was

upregulated upon K5-Cre mediated ablation of LAMA5, which was not seen in our

LAMC1EKO mice. In skin, transplantation experiments, where laminin-511 deficient

skin was transplanted onto full-thickness WT skin, resulted in a failure of hair germ

elongation leading to regression of HFs [25]. This regression of HFs, however, was

not observed in LAMC1EKO skin. Similar to our findings with LAMC1EKO skin,

embryonic laminin α5 deficient mice showed disrupted lamina densa and reduced

proliferation of hair matrix cells. Topically application of laminin-511 in the transplant

experiments, however, rescued these defects and also restored HF development

[25]. Laminin-511 in signaling during early hair germ elongation phase was implicated

by the reduced expression of the molecules sonic hedgehog (Shh) and Gli1 [25]. Our

gene expression analysis did not reveal any reduction in molecules of the Shh/Gli1

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signaling pathway at E18.5 and P6. Resembling the findings in our LAMC1EKO mice,

K5-mediated deletion of β1 integrin also lead to markedly reduced proliferation of

matrix keratinocytes and to a disrupted lamina densa [53]. More severe, β1 integrin

deletion was responsible for impaired invagination of HFs and eventual hair loss [53],

[54]. Integrin α3 deficient animals also displayed changes in BM ultrastructure, but

their HF phenotype was less severely disturbed than that observed in the absence of

integrin β1 and laminin α5 as the animals showed progressive hair loss only as adults

[56], [55]. Interestingly, the deletion of integrin β1 in the dermis did not affect HF

morphogenesis, suggesting that laminin-511 interacts with β1 integrins at the surface

of epithelial rather than on dermal papilla cells [57].

Our results suggest that loss of laminin-511 due to epidermal depletion of laminin γ1

causes abnormal expression of different hair keratins during late HF morphogenesis,

resulting in disruption of normal hair formation. We show that this is associated with

changes in the BMP-MSX2-HOXC13-FOXN1 signaling axis, which seems to be

crucially implicated in regulating HS differentiation and matrix cell proliferation.

Microarray analyses demonstrated that gene regulation in the skin of LAMC1EKO mice

at E18.5 was not markedly altered, probably due to a long half-life of laminin γ1

protein in the skin. At P6, however, many genes encoding hair keratins and keratin-

associated proteins (KAPs) were downregulated. This was accompanied by a

downregulation of the transcription factors HoxC13, FoxN1 and FoxQ1, which are all

known to directly or indirectly regulate expression of hair keratins found in the cortex,

medulla and cuticle layers of the HS [42], [43], [44]. The transcription factors HoxC13

[42] and FoxN1 [58], [59] have been shown to be essential for proper HS

differentiation as the corresponding mutants completely lack external hair. It has

been described that HoxC13 and FoxN1 might function in a common pathway of HF

differentiation, where FoxN1 acts downstream of HoxC13 in a regulatory signaling

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cascade regulating expression of terminal differentiation markers [60]. HoxC13 was

also described to target FoxQ1 expression during regulation of HF differentiation [61].

Interestingly, there is a partial overlap of the regulated genes in HOXC13 deficient

mice [60] and our LAMC1EKO mice. Ten out of 19 significantly regulated keratins in

the skin of LAMC1EKO mice were also downregulated in skin of HoxC13 mutants: K26,

K31, K32, K33a, K33b, K34, K35, K72, K84 and K86 [60]. Consistent with these

findings, MSX2, which is known to regulate the expression of FOXN1 and also of hair

keratins by a parallel signaling pathway [62], was also downregulated in LAMC1EKO

skin. Bone morphogenetic proteins (BMPs) act upstream of HoxC13, FoxN1 and

Msx2, and regulate postnatal differentiation and proliferation of HFs. They are

expressed in proliferating hair matrix cells and differentiating hair precursor cells.

Expression of the BMP inhibitor Noggin, under the control of the Msx2 promoter lead

to downregulation of hair keratins as well as of FoxN1 and HoxC13 expression.

Along with the findings from LAMC1EKO mice, differentiation of the HS, but not of the

IRS was disturbed in Msx2-Noggin transgenic mice [23]. In LAMC1EKO skin, we also

detected reduced phospho-Smad1/5 (pSmad1/5) expression in the skin at P6 and

P10, demonstrating that BMP signaling is altered in the absence of laminin γ1.

It has been shown that activated BMP signaling keeps HF stem cells in a quiescent

state. However, when BMP signaling is turned off, stem cells begin to proliferate and

produce transient amplifying (TA) progenitors that express Shh. Subsequently, BMP

signaling promotes TA cells to specialize and differentiate into the HS or the IRS

[22]. Thus, BMP inhibitor Noggin expressed by the dermal papilla sustains

proliferation and specification of TA cells within the hair bulb [63], [64]. The model in

Fig. 6 summarizes all the signaling pathways that we found altered in LAMC1EKO hair

follicles and which contribute to HS differentiation. During hair differentiation, BMP

signaling induces a signaling cascade in which Smad1/5 proteins become

18

phosphorylated. Epithelial BMP, through phosphorylated Smad1/5 induces Msx2,

which in turn activates HoxC13. HoxC13 was shown to directly regulate the

expression of FoxN1 and of HS keratins, either directly by binding to keratin

promotors or via FoxN1 [60]. HoxC13 also targets FoxQ1 expression and thereby

regulates HF differentiation [61]. Msx2 can also regulate FoxN1 not only via HoxC13,

but also directly.

Lef1, which mediates nuclear responses to Wnt signaling, was also suggested to

play an activating role in hair maturation. It cooperates with BMPs to activate HS

differentiation and keratin expression [23]. Lef1 mutant mice show disturbed HF

morphogenesis with downregulation of K31 [20], which, interestingly, is also

significantly downregulated in the skin of LAMC1EKO mice (Fig. 4a). These similarities

support the hypothesis that downregulation of Lef1 contributes to the phenotype of

LAMC1EKO mice.

All these regulatory proteins involved in HS maturation, however, are downregulated

in the absence of laminin-511 and altered localization of laminin-211, thereby

resulting in a reduced proliferation of matrix cells and a disturbed expression of

differentiation specific HS keratins (Fig. 6).

Taken together, our data suggest that there is a direct link between laminin-511-

mediated signaling and the BMP-MSX2-HOXC13-FOXN1 signaling axis, which

controls the regulation of HS differentiation and proliferation of hair matrix cells.

19

Experimental ProceduresGeneration and genotyping of mice with keratinocyte specific deletion of the

laminin γ1 chain (LAMC1EKO)

To generate the K14-Cre/LAMC1 knockout mouse strain, homozygous floxed laminin

γ1 mice (LAMC1flox) [33] were crossed with male K14-Cre transgenic mice [32]. Both

mouse strains were back-crossed for at least 8 generations onto the C57BL6/J

background. To obtain knockout mice that carry homozygous LAMC1flox alleles and

the Cre-recombinase transgene (LAMC1EKO mice), homozygous LAMC1flox mice

were crossed with heterozygous LAMC1flox mice carrying the K14-Cre transgene.

Littermates that were used as control animals in all experiments were either

homozygous LAMC1flox or heterozygous for the LAMC1flox allele and the Cre

transgene. Genotyping was performed by PCR using genomic DNA from mouse tail

biopsies. Wild-type, floxed or deleted alleles of LAMC1 were assessed using the

following primer sets: for detection of wildtype allele 5’-

CTCAGAGCTGGCTTCTCACAT-3’ and 5’-CATTTCCCCACAAGTGGTTCTT-3’, for

detection of floxed and deleted allele 5’-CCTACATTTTGAATGCAAGGATTGG-3’ and

5’-GATTTTCAAAGAAGCAGAGTGTG-3’. Animals were housed in specific-pathogen-

free facilities and all animal experiments were performed in compliance with German

Regulations for Welfare of Laboratory Animals and were approved by the

Regierungspräsidium Köln Germany (NRW authorization 8.87-51.05.20.13.004).

Histological analysis and indirect IF staining of skin sections

For histology, back skin samples were fixed in 4% (w/v) paraformaldehyde-

phosphate-buffered saline on ice for 45 min, embedded in paraffin wax and sectioned

(30-60nm thickness). Paraffin sections were subjected to trypsin digestion or citric

acid-based antigen retrieval. Alternatively, skin tissue was snap-frozen in optimal-

20

cutting-temperature compound (OCT Tissue Tec). Frozen sections were fixed with

ice-cold 100% ethanol or acetone. To block unspecific binging, skin sections were

incubated for one hour with 10% (v/v) donkey or goat serum in phosphate-buffered

saline, then incubated with the primary antibody, either for one hour at room

temperature or overnight at 4°C. Bound antibodies were detected with Alexa Fluor

coupled secondary antibodies. For visualization of nuclei, sections were stained with

DAPI. IF stainings were documented with the Olympus IX71 microscope. Information

on antibodies used is provided in the supplementary methods.

Hematoxylin and eosin staining of frozen skin sections was performed, according to

standard protocols. Light microscopy was performed with the Leica DM 4000 D

microscope.

Immunoblot analysis

Proteins of cell or total skin lysates from control and LAMC1EKO mice at P1 or

precipitates of cell culture supernatants (50µg) were resolved in 4-12% SDS-

polyacrylamide gradient gels (Invitrogen) under reducing conditions and transferred

to nitrocellulose membranes (GE Healthcare). The membranes were blocked in 5%

low-fat milk powder in TRIS-buffered saline and incubated with the respective

primary antibodies, followed by incubation with appropriate secondary antibodies

conjugated to horseradish peroxidase. Reactive bands were visualized using ECL

detection reagent (GE Healthcare) on X-ray films. Equal protein loading was

assessed by incubating the membranes with antibodies directed against GAPDH or

by Ponceau S staining. Detailed information on antibodies used is provided in the

supplementary methods.

Electron microscopy

21

Briefly, specimens were fixed in 2% glutaraldehyde/2% (w/v) paraformaldehyde (in

0.1M cacodylate buffer pH 7.4), followed by incubation in 2% OsO4/0.1M cacodylate

buffer, pH 7.4. After washing, the specimens were stained in 1% uranyl acetate,

dehydrated through a graded series of ethanol, and embedded in araldite resin.

Semithin sections (1μm) and ultrathin sections (70nm) were cut with a diamond knife

using an ultramicrotome (Reichert, Bensheim, Germany).

Isolation and cultivation of primary keratinocytes

Keratinocytes were isolated between postnatal day 1 and 2. The epidermis was

removed en bloc after Dispase treatment overnight (5mg/ml in serum-free DMEM) at

4°C. The epidermis was then incubated in a Trypsin substitute TrypLETM Select

(Gibco) for 40 min at room temperature. Afterwards, epidermal cells were dissolved

in serum-free DMEM and resuspended in low Ca2+ FAD medium. Low Ca2+ FAD

medium contains 10% (v/v) fetal calf serum (FCS) treated with Chelex 100 resin

(BioRad, Munich, Germany), adenine (1.8x10–10M), hydrocortisone (0.5µg/ml), insulin

(5g/ml), cholera toxin (10–10M), EGF (10ng/ml), streptomycin/penicillin (100U/ml) and

glutamine (0,1mg/ml). 106 cells were plated onto 6cm collagen type I-coated dishes

(BioCoat, BD) together with Mitomycin C-treated 3T3 fibroblasts. Information on

antibodies used is provided in the supplementary methods.

Statistical analysis

Statistical analyses of the data was performed using Mann-Whitney-Test. Differences

with P<0.05 were considered to be statistically significant (* p<0.05, ** p<0.005).

22

Acknowledgements

We thank Marion Reibetanz for expert technical assistance, and Dr. Ulrike Mayer

(University of Norwich) for stimulating discussion. We also thank Dr. Sidney

Strickland (Rockefeller University, New York, USA) for kindly providing the floxed

laminin γ1 strain. This work was supported by Deutsche Forschungsgemeinschaft

through SFB 829 (to WB, PZ, TK, BE, RN) at the University of Cologne and SFB

1009 (to LS) at the University of Muenster.

23

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26

Figure legendsFigure 1. Loss of laminin-511 deposition due to depletion of laminin γ1 in basal

keratinocytes leads to changes in laminin composition and to ultrastructural

defects of the DEJ. (a) LAMC1EKO mice show retarded HF morphogenesis, delayed

pigmentation and syndactyly at the hind limbs, when compared with control

littermates (P1, P3, P6, P10). Representative pictures are shown. (b) Representative

IF stainings of back skin sections of control and LAMC1EKO mice (E18.5, P1, P10)

using antibodies against the laminin γ1, α5 and α2 chains. The DEJ of LAMC1EKO

mice shows reduced deposition of laminin γ1 (arrow), loss of laminin α5 (arrowhead)

and ectopic deposition of α2 (asterisk). Scale bar: E18.5: 25µm; P1, P10: 50µm; n=5-

8. (c) Immunoblot analysis of keratinocyte lysates (50µg protein) and protein

precipitates from 0.5ml serum-free supernatants of keratinocytes and fibroblasts

isolated from control or LAMC1EKO skin. Primary cells were cultivated from four mice

per genotype at P1. Keratinocytes and fibroblasts were starved for 48h and 24h,

respectively. Ponceau S staining or GAPDH were used as loading control. (d)

Ultrastructure of control and mutant skin sections (P1 and P9). Asterisk: less tightly

packed basal keratinocytes; arrow: areas with interrupted lamina densa between

hemidesmosomes. Insets: three-fold higher magnification of the BM zone. E:

epidermis; D: dermis. Scale bar: 1µm (P1), and 5µm (P9).

Figure 2. HF morphogenesis is delayed in mice lacking epidermal laminin γ1

expression. (a) H&E stainings of paraffin-embedded back skin sections of control

and mutant mice at P1 and P10. Arrow: curved and inappropriately angled HFs;

arrowhead: barely formed HS fragments; asterisks: thickening of the epidermis

around HF regions. Scale bar: 50µm. (b) Ultrastructure of the follicular BM

surrounding the dermal papilla in HFs of control and mutant mice (P3). Arrow:

thickening of the BM; arrowhead: intact areas of the BM. Scale bar: 1µm. (c)

27

Categorization of HFs at P1 and P10 according to characteristics of HF

morphogenesis stages 0-8 as described (below). Quantification of total number (%)

of HFs for each developmental stage; means±SEM; P1: n=5-6; P10: n=5. (d)

Immunohistochemical staining of paraffin sections for Ki67 positive hair matrix cells

(brown) in control and LAMC1EKO skin (P10). Scale bar: 25µm. (e) Quantification of

proliferating matrix cells below Auber’s line (red line) as a percent to the total number

of matrix cells; means±SEM; n=6.

Figure 3. Early embryonic HF development is not disturbed in skin of LAMC1EKO

mice. (a) Categorization of embryonic HFs at day 16.5. Counting of total numbers

(%) of HF precursors in placode, peg or germ stages; means±SD; n=4. (b) IF

stainings of back skin cryosections from control and mutant mice at E16.5. Dotted

line: polarized area for P-cadherin expression, marks anterior side of HFs. Scale bar:

20µm (one bar for all). (c) Quantification of placode numbers in E16.5 mice after IF

staining for P-cadherin. 6-9 microscopic fields per animal were analyzed; means±SD,

n=4. (d) Quantification of HF numbers which end in the epidermis in 8-week old mice

under bright field microscopy after H&E staining. 4-5 microscopic fields per animal

were analyzed; means±SD, n=5-6.

Figure 4. Altered differentiation of the HS in LAMC1EKO mice. (a) Partial list of

significantly downregulated HS keratins in the epidermis and dermis of male

LAMC1EKO skin at P6 compared to control male mice as obtained by microarray

analysis (>1.5 fold, p<0.05); n=2. Results were validated by IF; n=3 (b) IF stainings of

frozen sections of control and LAMC1EKO skin (P6) using different hair keratin

antibodies (green). Development of the companion layer (K75) and the IRS (K28) of

control and LAMC1EKO HFs were comparable. In LAMC1EKO mice, differentiation of

hair cuticle (K82, K85), cortex (K33, K35, K85, K86) and medulla (K81) of the HS

28

were abnormal compared to control mice. Co-staining with K14 antibody (red). Nuclei

(DAPI; blue). Scale bar: 20µm (one bar for all).

Figure 5. Downregulated expression and impaired localization of transcription

factors regulating differentiation of hair specific keratin genes. (a) Summarized

list of significantly down-regulated transcription factors and regulatory proteins at P6

in the epidermis and dermis of LAMC1EKO skin compared to control mice identified by

RNA microarray analysis. (b) IF stainings of paraffin sections of skin from control and

mutant mice (P1, P6 and P10) using antibodies against FoxN1 and HoxC13. Nuclei

(DAPI; blue). Insets: two-fold higher magnification. Scale bar: 20µm; n=4. (c)

Immunoblot analyses of FoxN1, HoxC13, Msx2 and (d) phosphorylated Smad1 and -

5 (pSmad1/5) protein in LAMC1EKO mice compared to control animals (50µg total skin

lysates; P6, P10). GAPDH: loading control; n=4.

Figure 6. Schematic model summarizing the activity of proteins in HF

morphogenesis that are downregulated in LAMC1EKO skin. Expression of HS

keratins during HF differentiation is mainly regulated by signaling molecules of the

BMP-MSX2-HOXC13-FOXN1 signaling axis. The loss of epidermal laminin-511

results in downregulation of this BMP signaling axis, which in turn results in reduced

expression of the HS keratins depicted in the model. Red arrows: downregulated in

LAMC1EKO skin. See text for additional discussion. DP: dermal papilla, ORS: outer

root sheath, SCs: stem cells.

29